Latest revision as of 22:48, 1 November 2017

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Our research work

Research work

Finding a suitable topic was very challenging and time consuming. Initially, we looked through projects of prior teams and assembled a list of possible topics.

A big influence was a new method for assembling genes in a manufacturing manner which was being developed by a research group on our university. Based on the quick and easy synthesis of proteins a first idea was the creation of enzymes that could convert blood groups. Also working with cyanobacteria was an option we considered.

After many seminars, we established the idea of metabolic channeling using dCas9 as our main project. One of our advisors also worked with membraneless organelles and suggested this approach for achieving metabolic channeling and therefor our secondary project with LLPS.

We thought about using either violacein or beta carotene as exemplary pathways for our increased production but finally decided for beta carotene. This brought many new challenges in the form of understanding the pathway and implementing it in E. coli.

Also, we very worried that an increased output would end up consuming too much precursor substrate and hinder growth of the transformed cells. Additionally, we found that team Edinburgh/Glasgow had problems with toxicity if the enzymes of the beta carotene pathway were in a specific order.

But all the planning was for nothing when we realized that some of the enzymes of the beta carotene pathway were localized in the membrane and therefore not suitable for our metabolic channeling approach.

After planning the design more precise we eventually arrived at our scaffold design of a low and a high-copy plasmid.

Protocols

1. Preparation of starting plasmids

1. Preparation restriction of components

1. Restriction using biobrick assembly enzymes.
2. This preparation step is needed to create sticky ends on the cassettes.
3. This step is only performed once.
4. Restriction with EcoRI and PstI (see restriction protocol) for all components.

2. Ligation of cassettes into plasmids

1. Ligation using T4-ligase (see ligation protocol)

plasmid	cassettes
psB1C3	1	2	3
psB1A3	1	2	3
psB1K3	1	2	3

3. Final preparation steps

1. Transformation of 9 different combinations into competent cells (see transformation protocol).
2. Selection with corresponding antibiotics.

4. Next day

1. Colony pcr and gel run to check for sizes of cassettes.
2. Miniprep and check concentration via nanodrop.
3. Many aliquots needed (small volume because thawing time) for future reactions!

2. 3A-assembly

1. Good to know

1. The 3-A-assembly will be used to add more and more cassettes in a row.
2. It is important to only combine cassettes with the same number (1, 2 and 3 have varying spacer length).
3. We will add cassettes and test frequently for viability to determine the maximum target-sequence length.
4. We want to combine about five cassettes.

2. Assembly scheme

1. In each assembly cycle, there will be two cassettes (same number/length) added to one linearized plasmid.
2. In the first step, we can either just use the prepared plasmid with cassettes already inserted or use an empty one, because the part in between will be cut out anyway.
3. The be able to select for plasmid with higher cassette content the resistances will cycle.
4. The resistance cycle (for plasmids) is K C A.
5. For the inserts, the resistance signals from which plasmid they will be cut.
6. The plasmids resistance determines the selection antibiotics for that step!

3. Legend

CX – Chloramphenicol (Insert/Plasmid) from step X
AX – Ampicillin (Insert/Plasmid) from step X
KX – Kanamycin (Insert/Plasmid) from step X
E – EcoRI
S – SpeI
X – XbaI
P – PstI

Insert 1 E+S	Insert 2 X+P	Plasmid E+P
C1	A1	K1
A2	K1	C2
K3	C2	A3
C4	A3	K4

4. Starting a cycle

1. Insert 1 is cut out of plasmid C1/Insert 2 is cut out of plasmid A1 and plamsid K1 is linearized .
2. Insert 1 and 2 are ligated into plasmid K1.
3. Insert 1 is cut out of plasmid A2/Insert 2 (here the fusion from Insert 1+2 from the first cycle) is cut out of K1 and plasmid C2 is linearized.
4. Insert 1 and 2 are ligated into plasmid C2.
5. With insert 1 coming from K3 and insert 2 from C2 (fusion) the steps will repeated until maximum number of inserts is reached.
6. The assembly needs to be done for all 3 cassettes simultaneously after each step.
7. Transformation of ligation into competent cells (see transformation protocol).
8. Selection with corresponding antibiotics.

5. Next day

1. Colony pcr and gel run to check for sizes of cassettes.
2. Miniprep and check concentration via nanodrop.

^[1] http://parts.igem.org/Help:Protocols/3A_Assembly (accessed 31 October 2017)

1. Aim

1. Is the insert DNA in the plasmid present or absent?

2. Much easier than to isolate and purify the vector.

2. Good to know before the start

1. Take typical measures to prevent PCR cross over contamination, keep your bench clean, wear gloves, use sterile tubes and filter pipet tips.
2. Include a no-template control and positive control in parallel.
3. Thaw and keep reagents on ice.
4. Mix well before use.
5. The longer the amplicon, the longer the extension time: Use 15 sec/kb extension.
6. Use 90 sec extension for multiplexing.
7. Run an annealing temperature gradient from 55 °C to 65 °C to choose the best specificity conditions. Do not use fast cycling for multiplexing.
8. ALLin™ Red Taq Mastermix, 2X is premixed with red dye and density reagents for direct loading on the gels after the PCR. In a 2 % agarose TAE gel the dye migrates with ~350 bp DNA, in 1 % agarose TAE gel with ~600 bp DNA fragments.

3. Are you working with A. E.coli or B. yeast?

A.step by step for E.coli:

1. Resuspend colonies.

2. Prepare masterplate.

3. Prepare a PCR master mix (always prepare at least 10 % more).

4. Mix gently, avoid bubbles.

5. Aliquote the 22.0 μl of PCR master mix into each PCR tube.

6. Pick colony and put the toothpick or tip one into the Masterplate and then in the PCR tube/mix.

7. Do not forget the negative control!

8. Close tube.

9. Perform the PCR using Thermocycler as follow:

Steps	Cycles	Temperature	Time
Initial denaturation	1 cycle	95°C	60s
Denaturation	30-40 cycles	95°C	15s
Annealing	30-40 cycles	55-65°C	15s
Extension	30-40 cycles	72°C	15-90s (15 sec per 1 kb)
Final extension	1 cycle	72°C	5 min

10. Store probes for short time on ice, for long at -20 °C.

11. Load probes on the agarose gel e.g. 10 μl (so in case you have enough left for another round).

B. Step by step for yeast:

1. If resuspended colonies are to be used: pipette 50 μl of a 0.02 M NaOH solution into each of a set of appropriately labelled PCR tubes or wells of a PCR plate. Using sterile pipette tips or toothpicks, transfer transformants to individual tubes/wells. The amount of cells resuspended must just be visible. Resuspend cells by pipetting or vortexing and incubate for ≥ 5 min at 37 °C.
2. If overnight cultures are to be used: pipette 40 μl of a 0.01 M NaOH solution into each of a set of appropriately labelled PCR tubes or wells of a PCR plate. Transfer 10 μl of each overnight culture to be tested to the appropriate tube/well and mix by pipetting up and down. Incubate for ≥ 5 min at 37 °C.
3. Prepare a PCR master mix (always prepare at least 10 % more).
4. Aliquot 22.5 μl of PCR master mix into each PCR tube.
5. Add 2.5 μl of the resuspended colony or overnight culture mixed with NaOH to the appropriate PCR tube.
6. Close the tubes
7. Perform the PCR using the following cycling profile:

Steps	Cycles	Temperature	Time
Initial denaturation	1 cycle	95°C	60s
Denaturation	30-40 cycles	95°C	15s
Annealing	30-40 cycles	55-65°C	15s
Extension	30-40 cycles	72°C	15-90s
Final extension	1 cycle	72°C	5 min

8. Load probes on the agarose gel.

9. Store probes for short time on ice, for long at -20°C.

^{[1]https://www.highqu.com/media/wysiwyg/ressources/manuals/PCM02_ALLin_Red_Taq_Mastermix_PI.pdf (accessed 31 October 2017)}

1. What is it ?

1. Standard lab procedure for separating DNA by size (e.g., length in base pairs) for visualization and purification.
2. Uses an electrical field to move the negatively charged DNA through an agarose gel matrix toward a positive electrode.
3. Shorter DNA fragments migrate through the gel more quickly than longer ones.

2. Why are we doing it ?

1. to determine the approximate length of a DNA fragment by running it on an agarose gel alongside a DNA ladder (a collection of DNA fragments of known lengths).

3. Protocol

1. Pouring a Standard 1% Agarose Gel:

1. Measure 1g agarose and and mix it with 100 ml of TBE in a microwaveable flask.
Note: Agarose gels are commonly used in concentrations of 0.7 % to 2 % depending on the size of bands needed to be separated - Simply adjust the mass of agarose in a given volume to make gels of other agarose concentrations (e.g., 2 g of agarose in 100 mL of TAE will make a 2 % gel).
2. Microwave for 1-3 min until the agarose is completely dissolved (but do not overboil the solution, as some of the buffer will evaporate and thus alter the final percentage of agarose in the gel. Many people prefer to microwave in pulses, swirling the flask occasionally as the solution heats up.).
Note: gloves and glasses ! Caution HOT! Be careful stirring, eruptive boiling can occur.
It is a good idea to microwave for 30-45 sec, stop and swirl, and then continue towards a boil. Keep an eye on it as the initial boil has a tendency to boil over. Placing saran wrap over the top of the flask can help with this, but is not necessary if you pay close attention.

4. Pouring of the gel

1. Let agarose solution cool down to about 50 °C (about when you can comfortably keep your hand on the flask), about 5 mins.
Note: Or cool down in water bath about 30 min.
2. Add ethidium bromide (EtBr) to a final concentration of approximately 0.2-0.5 μg/mL (usually about 2-3 μl of lab stock solution per 100 mL gel). EtBr binds to the DNA and allows you to visualize the DNA under ultraviolet (UV) light.
Note: Caution EtBr is a known mutagen. Wear a lab coat, eye protection and gloves when working with this chemical. If you add EtBr to your gel, you will also want to add it to the running buffer when you run the gel.
3. Pour the agarose into a gel tray with the well comb in place.
Note: Think about witch gel tray size you need (a small one or a big one).
Pour slowly to avoid bubbles which will disrupt the gel. Any bubbles can be pushed away from the well comb or towards the sides/edges of the gel with a pipette trip.
4. Let the newly poured gel sit at room temperature for 20-30 mins, until it has completely solidified.
if you are in a hurry the gel can also be set more quickly if you place the gel tray at 4 °C earlier so that it is already cold when the gel is poured into it.

5. Loading Samples and Running an Agarose Gel

1. Add loading buffer to each of your digest samples.
Note: Loading buffer serves two purposes: 1) it provides a visible dye that helps with gel loading and will also allows you to gauge how far the gel has run while you are running your gel; and 2) it contains a high percentage of glycerol, so it increases the density of your DNA sample causing it settle to the bottom of the gel well, instead of diffusing in the buffer.
2. Once solidified, place the agarose gel into the gel box (electrophoresis unit).
3. Fill gel box with 1xTAE (or TBE) until the gel is covered.
4. Carefully load a molecular weight ladder into the first lane of the gel.
Note: When loading the sample in the well, maintain positive pressure on the sample to prevent bubbles or buffer from entering the tip. Place the very top of the tip of the pipette into the buffer just above the well. Very slowly and steadily, push the sample out and watch as the sample fills the well. After all of the sample is unloaded, push the pipettor to the second stop and carefully raising the pipette straight out of the buffer.
5. Carefully load your samples into the additional wells of the gel.
6. Run the gel at 80-150 V until the dye line is approximately 75-80 % of the way down the gel.
Note: Black is negative, red is positive. (The DNA is negatively charged and will run towards the positive electrode.) Always Run to Red.
Note: A typical run time is about 1-1.5 hours, depending on the gel concentration and voltage.
7. Turn OFF power, disconnect the electrodes from the power source, and then carefully remove the gel from the gel box.
8. Using any device that has UV light, visualize your DNA fragments.
Note: When using UV light, protect your skin by wearing safety goggles or a face shield, gloves and a lab coat.
Note: If you will be purifying the DNA for later use, use long-wavelength UV and expose for as little time as possible to minimize damage to the DNA.
Note: The fragments of DNA are usually referred to as ‘bands’ due to their appearance on the gel.

6. Analyzing Your Gel

Using the DNA ladder in the first lane as a guide (the manufacturer's instruction will tell you the size of each band), you can interpret the bands that you get in your sample lanes to determine if the resulting DNA bands that you see are as expected or not. For more details on doing diagnostic digests and how to interpret them please see the Diagnostic Digest page.

7. Purifying DNA from Your Gel

If you are conducting certain procedures, such as molecular cloning, you will need to purify the DNA away from the agarose gel. For instructions on how to do this, visit the Gel Purification page.

^{[1]
http://www.addgene.org/protocols/gel-electrophoresis/ (accessed 31 October 2017)}

1. Aim

Isolation of DNA as a plasmid

2.Production of cleared lysate

1. Isolation of the bacteria

1. Harvest 1–5 ml (high-copy-number plasmid) or 10 ml (low-copy-number plasmid) of bacterial culture .
2. Centrifugation for 5 minutes at 10,000 xg in a tabletop centrifuge.
3. Pour off the supernatant.
4. Reinsert again bacterial culture to the pellet and repeat step 2 and 3.
5. Blot the inverted tube on a paper towel to remove excess media.

2. Resuspension of the cells

1. Add 250 μl of Cell Resuspension Solution.
2. Completely resuspend the cell pellet by vortexing or pipetting.
3. It is essential to thoroughly resuspend the cells.

3. Lysing

1. Add 250 μl of Cell Lysis Solution.
2. Mix by inverting the tube 4 times - do not vortex.
3. Incubate until the cell suspension clears (clear ≠ colorlessly) (approximately 1–5 minutes).

4. Splitting proteins

1. Add 10 μl of Alkaline Protease Solution.
2. Mix by inverting the tube 4 times - do not vortex.
3. Incubate for 5 minutes at room temperature.

5. Neutralization

1. Add 350 μl of Neutralization Solution.
2. Immediately mix by inverting the tube 4 times - do not vortex.

6. Isolation of the plasmids

1. Centrifuge the bacterial lysate at maximum speed (around 14,000 ×g) in a microcentrifuge for 10 minutes at room temperature.

3. Isolation of the plasmid DNA

1. Transfer the cleared lysate (approximately 850 μl, Section 3.B, Step 6) to the prepared Spin Column by decanting. Avoid disturbing or transferring any of the white precipitate with the supernatant.

1. If the white precipitate is accidentally transferred to the Spin Column, pour the Spin Column contents back into a sterile 1.5ml microcentrifuge tube and centrifuge for another 5–10 minutes at maximum speed. Transfer the resulting supernatant into the same Spin Column that was used initially for this sample. The Spin Column can be reused but only for this sample.

2. Centrifuge the supernatant at maximum speed in a microcentrifuge for 1 minute at room temperature. Remove the Spin Column from the tube and discard the flowthrough from the Collection Tube. Reinsert the Spin Column into the Collection Tube.

3. Wash the plasmid DNA.

1. Add 750 μl of Column Wash Solution.
2. Centrifuge at maximum speed in a microcentrifuge for 1 minute at room temperature.
3. Remove the Spin Column from the tube and discard the flowthrough.
4. Reinsert the Spin Column into the Collection Tube.

4. Wash again the plasmid DNA.

1. Add 250 μl of Column Wash Solution.
2. Centrifuge at maximum speed in a microcentrifuge for 2 minutes at room temperature.
3. If the Spin Column has Column Wash Solution associated with it, centrifuge again for 1 minute at maximum speed.
4. Transfer the Spin Column to a new, sterile 1.5ml microcentrifuge tube, being careful not to transfer any of the Column Wash Solution with the Spin Column.

5. Elute the plasmid DNA

1. Add 50 μl of Nuclease-Free Water to the Spin Column, wait 5 minutes
2. Centrifuge at maximum speed for 1 minute at room temperature in a microcentrifuge.

6. After eluting the DNA, remove the assembly from the 1.5ml microcentrifuge tube and discard the Spin Column.

^[1]https://www.promega.de/-/media/files/resources/protocols/technical-bulletins/0/wizard-plus-sv-minipreps-dna-purification-system-protocol.pdf (accessed 31 October 2017)

1. Aim

A restriction digest is the division of DNA in a specific area by the help of restriction enzymes. The aim afterwards could be to analyze and characterize (restriction maps) the DNA, to compare it to others or to clone it into e.g. a vector. Previous to the restriction, the DNA has to be isolated (see protocol miniprep).

2. Test digest - What for?

In a test digest the cleavage products are analyzed to verify that the used DNA has e.g. taken in a specific fragment. Only few of the DNA is digested, because the uncut DNA will be used in further steps. The whole DNA strand is often too long to analyze, therefore:

targeted cuts between specific base sequences
cleavage fragments small enough to run on gel
segregation and analysis by gel electrophoresis

→ always with a control: uncut plasmid (see protocol gel electrophoresis) In a preparative digest normally 1 U enzyme digests 1 µg DNA in one hour.

3. Preparative digest - What for?

In a preparative digest the entire available DNA is digested, because the cleavage products are used in further steps. The cut DNA can be extracted from a gel. It is important that as much DNA as possible is digested. Therefore 0,2 - 0,4 µl enzyme per µg DNA are applied and the reaction should run for ~2h.

4. Procedure

exemplary pipetting scheme:

component	volume
DNA (typically 200 - 500 ng)	1 µl
10x buffer	1 µl
H₂O	7,6 µl
enzyme 1	0,1 µl
enzyme 2	0,1 µl
final volume	10 µl

If more than one reaction is done, it is convenient to prepare a master mix containing everything except for the DNA (!). This saves time and tips and keeps you from having to pipet very small volumes (e.g. 0,1 µl).
It is also possible to use more than two or just one restriction enzyme per reaction. They just need to have the same buffer preferences. Volumes have to be adjusted to the number of enzymes.

^[1] http://www.log2embl.de/sites/default/files/Labor-Protokoll-Restriktionsverdau.pdf (accessed 30 October 2017)

^[2] https://www.uni-hohenheim.de/fileadmin/einrichtungen/pflanzenphysiologie/Protokolle/V.Klonierung/restriktionsverdau_de.pdf (accessed 30 October 2017)

^[3] http://www.biochemie.uni-jena.de/files/Praktikum/plasmid%20dna%20+%20restrictionsverdau.pdf (accessed 30 October 2017)


If there is more than the wanted DNA band: A. Dissolving the Gel Slice	If there iss only one DNA band: B. Processing PCR Amplifications
1. Following electrophoresis, excise DNA band from gel (with scalpel) and place gel slice in a 1.5 ml microcentrifuge tube. 2. Add 10 μl Membrane Binding Solution per 10 mg of gel slice. Vortex and incubate at 50 – 65 °C until gel slice is completely dissolved.	You can just work with the rest of your PCR aliquote (when you did not use all of it for the gel electrophoresis). 1. Add an equal volume (so you have to know how much aliquote is still in your eppi!) of Membrane Binding Solution to the PCR amplification.

component	volume
T4 DNA Ligase Buffer (10 x)	2 µl
10x buffer	1 µl
T4 DNA Ligase	1 µl
Vector DNA
Insert DNA
Nuclease-free water	to 20 µl

Component	25 μl Reaction	50 μl Reaction	Final Concentration
Q5 High-Fidelity 2X Master Mix	12.5 μl	25 μl	1X
10 μM Forward Primer	1.25 μl	2.5 μl	0.5 μM
10 μM Reverse Primer	1.25 μl	2.5 μl	0.5 μM
Template DNA	variable	variable	< 1,000 ng
Nuclease-Free Water	to 25 μl	to 50 μl

Step	Temperature	Time
Initial Denaturation	98°C	30 seconds
	98°C	5–10 seconds
25–35 Cycles	*50–72°C	10–30 seconds
	72°C	20–30 seconds/kb
Final Extension	72°C	2 minutes
Hold	4-10°C

Difference between revisions of "Team:Potsdam/Protocols"

Latest revision as of 22:48, 1 November 2017

IGEM Potsdam

Research work

@@ Line 10: / Line 10: @@
          <div id="main" class="container">
              <div id="content">
-<p> We are describing our research work. Below you can find the protocols we used. </p>
+<h1>Research work</h1>
+<p align="justify">
+<br>
+Finding a suitable topic was very challenging and time consuming. Initially, we looked through projects of prior teams and assembled a list of possible topics.
+<br>
+<br>
+A big influence was a new method for assembling genes in a manufacturing manner which was being developed by a research group on our university. Based on the quick and easy synthesis of proteins a first idea was the creation of enzymes that could convert blood groups. Also working with cyanobacteria was an option we considered.
+<br>
+<br>
+After many seminars, we established the idea of metabolic channeling using dCas9 as our main project. One of our advisors also worked with membraneless organelles and suggested this approach for achieving metabolic channeling and therefor our secondary project with LLPS.
+<br>
+<br>
+We thought about using either violacein or beta carotene as exemplary pathways for our increased production but finally decided for beta carotene. This brought many new challenges in the form of understanding the pathway and implementing it in <i>E. coli</i>.
+<br>
+<br>
+<img src="https://static.igem.org/mediawiki/2017/8/85/T--Potsdam--home--enzyme.png" width="40%" style="float:right">
+Also, we very worried that an increased output would end up consuming too much precursor substrate and hinder growth of the transformed cells. Additionally, we found that team Edinburgh/Glasgow had problems with toxicity if the enzymes of the beta carotene pathway were in a specific order.
+<br>
+<br>
+But all the planning was for nothing when we realized that some of the enzymes of the beta carotene pathway were localized in the membrane and therefore not suitable for our metabolic channeling approach.
+<br>
+<br>
+After planning the design more precise we eventually arrived at our scaffold design of a low and a high-copy plasmid.
+</p>
@@ Line 43: / Line 67: @@
 <div class="spoiler">
-<input type="button" style="height: 50px; width: 50%; BACKGROUND-COLOR: #3399FF; font-size:25; color:black;"  onclick="showSpoiler(this);" value="3 A Assembly" />
+<input type="button" style="height: 50px; width: 50%; BACKGROUND-COLOR: rgb(61,67,80); font-size:25; color:white;"  onclick="showSpoiler(this);" value="3-A-Assembly" />
 <div class="inner" style="display:none;">
 <br>
 <div>
-<b> 1) Preparation of starting plasmids </b>
+<b> 1. Preparation of starting plasmids </b>
 <br><br>
+<div style="text-align: justify; margin-left:20px">
+. Preparation restriction of components </div>
-<u>Preparation restriction of components:</u>
+<div style="text-align: justify; margin-left:40px">
-<br> <br>
+. Restriction using biobrick assembly enzymes. <br>
-<li><div style="text-indent:30px;"> restriction using biobrick assembly enzymes </li></div>
+. This preparation step is needed to create sticky ends on the cassettes. <br>
-<li><div style="text-indent:30px;"> this preparation step is needed to create sticky ends on the cassettes </li>
+. This step is only performed once. <br>
-<li> <div style="text-indent:30px;">this step is only performed once </li>
+. Restriction with EcoRI and PstI (see restriction protocol) for all components. </div>
-<li> <div style="text-indent:30px;">restriction with EcoRI and PstI (see restriction protocol) for all components </li>
-</u>
 <br>
-<u>Ligation of cassettes into plasmids:</u>
+<div style="text-align: justify; margin-left:20px">2. Ligation of cassettes into plasmids </div>
-<br> <br>
+<div style="text-align: justify; margin-left:40px">
-<li><div style="text-indent:30px;">ligation using T4-ligase (see ligation protocol)
+. Ligation using T4-ligase (see ligation protocol)</div>
-<br> <br>
+<br>
 <style type="text/css">
 .tg  {border-collapse:collapse;border-spacing:0;}
@@ Line 81: / Line 105: @@
    </tr>
    <tr>
-     <td class="tg-yw4l" align="center">psB1C3</td>
+     <td class="tg-yw4l" align="center"><b>psB1C3</b></td>
      <td class="tg-yw4l"align="center">1</td>
      <td class="tg-yw4l"align="center">2</td>
@@ Line 87: / Line 111: @@
    </tr>
    <tr>
-     <td class="tg-yw4l"align="center">psB1A3</td>
+     <td class="tg-yw4l"align="center"><b>psB1A3</b></td>
      <td class="tg-yw4l"align="center">1</td>
      <td class="tg-yw4l"align="center">2</td>
@@ Line 93: / Line 117: @@
    </tr>
    <tr>
-     <td class="tg-yw4l"align="center">psB1K3</td>
+     <td class="tg-yw4l"align="center"><b>psB1K3</b></td>
      <td class="tg-yw4l"align="center">1</td>
      <td class="tg-yw4l"align="center">2</td>
@@ Line 99: / Line 123: @@
    </tr>
 </table>
-<br> <br>
-<u>Final preparation steps</u>
-<br> <br>
-<li><div style="text-indent:30px;"> transformation of 9 different combinations into competent cells (see transformation protocol)</li>
-<li><div style="text-indent:30px;">selection with corresponding antibiotics</li>
+<div style="text-align: justify; margin-left:20px">3. Final preparation steps </div>
-<br> <br>
+<div style="text-align: justify; margin-left:40px">
-<u>Next day</u>
+. Transformation of 9 different combinations into competent cells (see transformation protocol).<br>
+. Selection with corresponding antibiotics.</div>
 <br>
-<li><div style="text-indent:30px;"> colony pcr and gel run to check for sizes of cassettes </li>
+<div style="text-align: justify; margin-left:20px">4. Next day</div>
+<div style="text-align: justify; margin-left:40px">
+. Colony pcr and gel run to check for sizes of cassettes. <br>
-<li><div style="text-indent:30px;"> miniprep and check concentration via nanodrop </li>
+. Miniprep and check concentration via nanodrop. <br>
-<li><div style="text-indent:30px;">v many aliquots needed (small volume because thawing time) for future reactions!</li>
+. Many aliquots needed (small volume because thawing time) for future reactions!</div>
 <br>
 <br>
-<b>3) 3A-assembly</b>
+<b>2. 3A-assembly</b>
 <br> <br>
-<li><div style="text-indent:30px;"> the 3-A-assembly will be used to add more and more cassettes in a row </li>
+<div style="text-indent:20px;">1. Good to know </div>
+<div style="text-align: justify; margin-left:40px">
+. The 3-A-assembly will be used to add more and more cassettes in a row. <br>
-<li><div style="text-indent:30px;"> it is important to only combine cassettes with the same number (1, 2 and 3 have varying spacer length)</li>
+. It is important to only combine cassettes with the same number (1, 2 and 3 have varying spacer length).<br>
-<li><div style="text-indent:30px;"> we will add cassettes and test frequently for viability to determine the maximum target-sequence length</li>
+. We will add cassettes and test frequently for viability to determine the maximum target-sequence length.<br>
-<li><div style="text-indent:30px;"> we want to combine about five cassettes </li>
+. We want to combine about five cassettes. </div>
+<br>
+<div style="text-indent:20px;">2. Assembly scheme</div>
-<u>Assembly scheme</u>
-<li><div style="text-indent:30px;"> in each assembly cycle, there will be two cassettes (same number/length) added to one linearized plasmid </li>
+<div style="text-align: justify; margin-left:40px">
+. In each assembly cycle, there will be two cassettes (same number/length) added to one linearized plasmid. <br>
-<li><div style="text-indent:30px;"> in the first step, we can either just use the prepared plasmid with cassettes already inserted or use an empty one, because the part in between will be cut out anyway </li>
+. In the first step, we can either just use the prepared plasmid with cassettes already inserted or use an empty one, because the part in between will be cut out anyway. <br>
-<li><div style="text-indent:30px;"> the be able to select for plasmid with higher cassette content the resistances will cycle</li>
+. The be able to select for plasmid with higher cassette content the resistances will cycle.<br>
-<li><div style="text-indent:30px;">the resistance cycle (for plasmids) is K C A </li>
+. The resistance cycle (for plasmids) is K C A. <br>
-<li><div style="text-indent:30px;"> for the inserts, the resistance signals from which plasmid they will be cut</li>
+. For the inserts, the resistance signals from which plasmid they will be cut.<br>
-<li><div style="text-indent:30px;"> the plasmids resistance determines the selection antibiotics for that step!</li>
+. The plasmids resistance determines the selection antibiotics for that step!</div>
+<br>
+<div style="text-indent:20px;">3. Legend </div>
+<div style="text-align: justify; margin-left:40px">
+CX – Chloramphenicol (Insert/Plasmid) from step X
+<br>
+AX – Ampicillin (Insert/Plasmid) from step X
+<br>
+KX – Kanamycin (Insert/Plasmid) from step X
+<br>
+E – EcoRI
+<br>
+S – SpeI
+<br>
+X – XbaI
+<br>
+P – PstI
+</div> <br>
+<style type="text/css">
+.tg  {border-collapse:collapse;border-spacing:0;}
+.tg td{font-family:Arial, sans-serif;font-size:14px;padding:10px 5px;border-style:solid;border-width:1px;overflow:hidden;word-break:normal;}
+.tg th{font-family:Arial, sans-serif;font-size:14px;font-weight:normal;padding:10px 5px;border-style:solid;border-width:1px;overflow:hidden;word-break:normal;}
+.tg .tg-yw4l{vertical-align:top}
+</style>
+<table class="tg">
+  <tr>
+    <th class="tg-yw4l"" align="center"><b>Insert 1 E+S</b></th>
+    <th class="tg-yw4l"" align="center"><b>Insert 2 X+P</b></th>
+    <th class="tg-yw4l"" align="center"><b>Plasmid E+P</b></th>
+  </tr>
+  <tr>
+    <td class="tg-yw4l"" align="center">C1</td>
+    <td class="tg-yw4l"" align="center">A1</td>
+    <td class="tg-yw4l"" align="center">K1</td>
+  </tr>
+  <tr>
+    <td class="tg-yw4l"" align="center">A2</td>
+    <td class="tg-yw4l"" align="center">K1</td>
+    <td class="tg-yw4l"" align="center">C2</td>
+  </tr>
+  <tr>
+    <td class="tg-yw4l"" align="center">K3</td>
+    <td class="tg-yw4l"" align="center">C2</td>
+    <td class="tg-yw4l"" align="center">A3</td>
+  </tr>
+  <tr>
+    <td class="tg-yw4l"" align="center">C4</td>
+    <td class="tg-yw4l"" align="center">A3</td>
+    <td class="tg-yw4l"" align="center">K4</td>
+  </tr>
+</table>
+<br>
+<div style="text-indent:20px;">4. Starting a cycle</div>
+<div style="text-align: justify; margin-left:40px">
+. Insert 1 is cut out of plasmid C1/Insert 2 is cut out of plasmid A1 and plamsid K1 is linearized .<br>
-<hr size="10" noshade>
+. Insert 1 and 2 are ligated into plasmid K1. <br>
-<b>Legend</b>
+. Insert 1 is cut out of plasmid A2/Insert 2 (here the fusion from Insert 1+2 from the first cycle) is cut out of K1 and plasmid C2 is linearized. <br>
-<br> <br>
+. Insert 1 and 2 are ligated into plasmid C2.<br>
-<li> CX – Chloramphenicol (Insert/Plasmid) from step X
-<li> AX – Ampicillin (Insert/Plasmid) from step X
-<li> KX – Kanamycin (Insert/Plasmid) from step X
-<li> E – EcoRI
-<li> S – SpeI
-<li> X – XbaI
-<li> P – PstI
-<br> <br>
+. With insert 1 coming from K3 and insert 2 from C2 (fusion) the steps will repeated until maximum number of inserts is reached. <br>
+. The assembly needs to be done for all 3 cassettes simultaneously after each step.
 <br>
-  </div></div>
+. Transformation of ligation into competent cells (see transformation protocol).
+<br>
+. Selection with corresponding antibiotics. </div>
+<br>
+<div style="text-indent:20px;">5. Next day</div>
+<div style="text-align: justify; margin-left:40px">
+. Colony pcr and gel run to check for sizes of cassettes.
+<br>
+. Miniprep and check concentration via nanodrop.</div>
+<br> <br>
+<hr size="10" noshade></hr>
+<p style="font-size:10pt;"><sup>[1]</sup>
+http://parts.igem.org/Help:Protocols/3A_Assembly (accessed 31 October 2017)</p>
+  </div></div></div>
@@ Line 161: / Line 253: @@
 <div class="spoiler">
-<input type="button" style="height:50px; width:50%; BACKGROUND-COLOR: #3399FF; font-size:25; color:black;" onclick="showSpoiler(this);" value="Colony PCR" />
+<input type="button" style="height: 50px; width: 50%; BACKGROUND-COLOR: rgb(61,67,80); font-size:25; color:white;" onclick="showSpoiler(this);" value="Colony PCR" />
 <div class="inner" style="display:none;">
-<b>Colony PCR with ALLin™ Red Taq Mastermix, 2X: </b>
+<br>
+<div alignt="justify">
+<b>1. Aim </b><br><br>
+<div style="text-indent:40px;">
+. Is the insert DNA in the plasmid present or absent? </div>
+<div style="text-indent:40px;">
+. Much easier than to isolate and purify the vector. </div>
+<br>
+<b> 2. Good to know before the start </b> <br><br>
+<div style="text-align: justify; margin-left:20px">
+. Take typical measures to prevent PCR cross over contamination, keep your bench clean, wear gloves, use sterile tubes and filter pipet tips.
+<br>
+. Include a no-template control and positive control in parallel. <br>
+. Thaw and keep reagents on ice. <br>
+. Mix well before use.  <br>
+. The longer the amplicon, the longer the extension time: Use 15 sec/kb extension. <br>
+. Use 90 sec extension for multiplexing. <br>
+. Run an annealing temperature gradient from 55 °C to 65 °C to choose the best specificity conditions. Do not use fast cycling for multiplexing.  <br>
+. ALLin™ Red Taq Mastermix, 2X is premixed with red dye and density reagents for direct loading on the gels after the PCR. In a 2 % agarose TAE gel the dye migrates with ~350 bp  DNA, in 1 % agarose TAE gel with ~600 bp DNA fragments. </div>
 <br> <br>
-<b>aim: </b><br>
-Is the insert DNA in the plasmid present or absent? <br>
+<b>3. Are you working with <i> A. E.coli </i> or B. yeast?</b>
-Much easier than to isolate, purify the vector
-<br> <br>
-<b> good to know before the start: </b> <br>
- - Take typical measures to prevent PCR cross over contamination, keep your bench clean, wear gloves, use sterile tubes and filter pipet tips <br>
-- Include a no-template control and positive control in parallel. <br>
-- Thaw and keep reagents on ice <br>
-- Mix well before use.  <br>
-- The longer the amplicon, the longer the extension time: Use 15 sec/kb extension. <br>
-- Use 90 sec extension for multiplexing <br>
-- Run an annealing temperature gradient from 55 °C to 65 °C to choose the best specificity conditions. Do not use fast cycling for multiplexing.  <br>
-- ALLin™ Red Taq Mastermix, 2X is premixed with red dye and density reagents for direct loading on the gels after the PCR. In a 2% agarose TAE gel the dye migrates with~350 bp  DNA, in 1% agarose TAE gel with ~ 600 bp DNA fragments
 <br> <br>
-<b>step by step for E.coli: </b> <br> <br>
+<div style="text-indent:20px;">
-<div style="text-indent:10px;">- resuspend colonies: <br>
+<b>A.step by step for <i>E.coli</i>: </b> <br> <br>
-- Prepare masterplate
+<div style="text-indent:40px;">
-- Prepare a PCR master mix  (always prepare at least 10 % more, use the excel sheet by Sophia
+. Resuspend colonies. </div>
-to calculate) <br>
+<div style="text-indent:40px;">
-- 20 μl would be good, Fabian suggested 5 μl <br>
+. Prepare masterplate. </div>
-Mix gently, avoid bubbles.  <br>
+<div style="text-indent:40px;">
-- Aliquote the 22.0 μl of PCR master mix into each PCR tube. <br>
+. Prepare a PCR master mix  (always prepare at least 10 % more). </div>
-- Pick colony and put the toothpick or tip one into the Masterplate and then in the PCR tube/mix.  <br>
+<div style="text-indent:40px;">
-<br>
+. Mix gently, avoid bubbles.  </div>
-Do not forget the negative control! <br>
+<div style="text-indent:40px;">
-<br>
+. Aliquote the 22.0 μl of PCR master mix into each PCR tube. </div>
-- Close tube <br>
+<div style="text-indent:40px;">
-- Perform the PCR using Thermocycler as follow: <br>
+. Pick colony and put the toothpick or tip one into the Masterplate and then in the PCR tube/mix.  </div>
-<style type="text/css">
+<div style="text-indent:40px;">
-.tg  {border-collapse:collapse;border-spacing:0;}
+. Do not forget the negative control! </div>
-.tg td{font-family:Arial, sans-serif;font-size:14px;padding:10px 5px;border-style:solid;border-width:1px;overflow:hidden;word-break:normal;}
+<div style="text-indent:40px;">
-.tg th{font-family:Arial, sans-serif;font-size:14px;font-weight:normal;padding:10px 5px;border-style:solid;border-width:1px;overflow:hidden;word-break:normal;}
+. Close tube. </div>
-.tg .tg-yw4l{vertical-align:top}
+<div style="text-indent:40px;">
-</style>
+. Perform the PCR using Thermocycler as follow: </div>
-<table class="tg">
+<br>
+<table>
+<tr>
+    <th align="center"><b>Steps</b></th>
+    <th align="center"><b>Cycles</b></th>
+    <th align="center"><b>Temperature</b></th>
+    <th align="center"><b>Time</b></th>
+  </tr>
    <tr>
-     <th class="tg-031e"><b>Initial denaturation</b><br></th>
+     <th align="center"><b>Initial denaturation</b><br></th>
-     <th class="tg-yw4l"align="center">1 cycle<br></th>
+     <th align="center">1 cycle<br></th>
-     <th class="tg-yw4l"align="center">95°C</th>
+     <th align="center">95°C</th>
-     <th class="tg-yw4l"align="center">60s</th>
+     <th align="center">60s</th>
    </tr>
    <tr>
-     <td class="tg-yw4l"><b>Denaturation</b></td>
+     <td align="center"><b>Denaturation</b></td>
-     <td class="tg-yw4l"align="center">30-40 cycles<br></td>
+     <td align="center">30-40 cycles<br></td>
-     <td class="tg-yw4l"align="center">95°C</td>
+     <td align="center">95°C</td>
-     <td class="tg-yw4l"align="center">15s</td>
+     <td align="center">15s</td>
    </tr>
    <tr>
-     <td class="tg-yw4l"><b>Annealing</b></td>
+     <td align="center"><b>Annealing</b></td>
-     <td class="tg-yw4l"align="center">30-40 cycles<br></td>
+     <td align="center">30-40 cycles<br></td>
-     <td class="tg-yw4l"align="center">55-65°C</td>
+     <td align="center">55-65°C</td>
-     <td class="tg-yw4l"align="center">15s</td>
+     <td align="center">15s</td>
    </tr>
    <tr>
-     <td class="tg-yw4l"><b>Extension</b></td>
+     <td align="center"><b>Extension</b></td>
-     <td class="tg-yw4l"align="center">30-40 cycles<br></td>
+     <td align="center">30-40 cycles<br></td>
-     <td class="tg-yw4l"align="center">72°C</td>
+     <td align="center">72°C</td>
-     <td class="tg-yw4l"align="center">15-90s (15 sec per 1 kb)</td>
+     <td align="center">15-90s (15 sec per 1 kb)</td>
    </tr>
    <tr>
-     <td class="tg-yw4l"><b>Final extension</b></td>
+     <td  align="center"><b>Final extension</b></td>
-     <td class="tg-yw4l"align="center">1 cycle<br></td>
+     <td align="center">1 cycle<br></td>
-     <td class="tg-yw4l"align="center">72°C</td>
+     <td align="center">72°C</td>
-     <td class="tg-yw4l"align="center">5 min<br></td>
+     <td align="center">5 min<br></td>
    </tr>
 </table>
-<br> <br>
+<br>
-- Store probes for short time on ice, for long at -20°C <br>
+<div style="text-indent:40px;">
-- Load probes on the agarose gel e.g. 10 μl (so in case you have enough left for another round). <br>
+. Store probes for short time on ice, for long at -20 °C. </div>
+<div style="text-indent:40px;">
+. Load probes on the agarose gel e.g. 10 μl (so in case you have enough left for another round). </div>
 <br>
+<div style="text-indent:20px;">
+<b>B. Step by step  for yeast: </b></div>
+<div style="text-align: justify; margin-left:40px"><br>
+. If resuspended colonies are to be used: pipette 50 μl of a 0.02 M NaOH  solution into each of a set of  appropriately labelled PCR tubes or wells of a PCR plate. Using sterile pipette tips or toothpicks, transfer transformants to individual tubes/wells. The amount of cells  resuspended must just be  visible. Resuspend cells by pipetting or vortexing and incubate for
+≥ 5 min at 37 °C. <br>
+. If overnight cultures are to be used: pipette 40 μl of a 0.01 M NaOH solution into each of a set of appropriately labelled PCR tubes or wells of a PCR plate. Transfer 10 μl of each overnight culture to be tested to the appropriate tube/well and mix by pipetting up and down. Incubate for ≥ 5 min at 37 °C. <br>
-<b>step by step  for yeast: </b>
+. Prepare a PCR master mix (always prepare at least 10 % more).<br>
-- If resuspended colonies are to be used: pipette 50 μl of a 0.02 M NaOH  solution into each of a set of appropriately labelled PCR tubes or wells of a PCR plate. Using sterile pipette tips or toothpicks, transfer transformants to individual tubes/wells. The amount of cells
-resuspended must just be visible. Resuspend cells by pipetting or vortexing and incubate for
+. Aliquot 22.5 μl of PCR master mix into each PCR tube. <br>
-≥ 5 min at 37 °C. <br>
-If overnight cultures are to be used: pipette 40 μl of a 0.01 M NaOH solution into each of a set of appropriately labelled PCR tubes or wells of a PCR plate. Transfer 10 μl of each overnight culture to be tested to the appropriate tube/well and mix by pipetting up and down. Incubate for ≥ 5 min at 37 °C. <br>
+. Add 2.5 μl of the resuspended colony or overnight culture mixed with NaOH to the  appropriate PCR tube. <br>
-- Prepare a PCR master mix (always prepare at least 10% more, use the excel sheet to calculate)
-- Aliquot 22.5 μl of PCR master mix into each PCR tube. <br>
+. Close the tubes <br>
-- Add 2.5 μl of the resuspended colony or overnight culture mixed with NaOH to the  appropriate PCR tube. <br>
--  Close the tubes <br>
+. Perform the PCR using the following cycling profile: </div><br>
-- Perform the PCR using the following cycling profle: <br>
+<table>
-<style type="text/css">
+<tr>
-.tg  {border-collapse:collapse;border-spacing:0;}
+    <th align="center"><b>Steps</b></th>
-.tg td{font-family:Arial, sans-serif;font-size:14px;padding:10px 5px;border-style:solid;border-width:1px;overflow:hidden;word-break:normal;}
+    <th align="center"><b>Cycles</b></th>
-.tg th{font-family:Arial, sans-serif;font-size:14px;font-weight:normal;padding:10px 5px;border-style:solid;border-width:1px;overflow:hidden;word-break:normal;}
+    <th align="center"><b>Temperature</b></th>
-.tg .tg-yw4l{vertical-align:top}
+    <th align="center"><b>Time</b></th>
-</style>
+  </tr>
-<table class="tg">
    <tr>
-     <th class="tg-031e">Initial denaturation<br></th>
+     <th align="center"><b>Initial denaturation</b><br></th>
-     <th class="tg-yw4l">1 cycle<br></th>
+     <th align="center">1 cycle<br></th>
-     <th class="tg-yw4l">95°C</th>
+     <th align="center">95°C</th>
-     <th class="tg-yw4l">60s</th>
+     <th align="center">60s</th>
    </tr>
    <tr>
-     <td class="tg-yw4l">Denaturation</td>
+     <td align="center"><b>Denaturation</b></td>
-     <td class="tg-yw4l">30-40 cycles<br></td>
+     <td align="center">30-40 cycles<br></td>
-     <td class="tg-yw4l">95°C</td>
+     <td align="center">95°C</td>
-     <td class="tg-yw4l">15s</td>
+     <td align="center">15s</td>
    </tr>
    <tr>
-     <td class="tg-yw4l">Annealing</td>
+     <td align="center"><b>Annealing</b></td>
-     <td class="tg-yw4l">30-40 cycles<br></td>
+     <td align="center">30-40 cycles<br></td>
-     <td class="tg-yw4l">55-65°C</td>
+     <td align="center">55-65°C</td>
-     <td class="tg-yw4l">15s</td>
+     <td align="center">15s</td>
    </tr>
    <tr>
-     <td class="tg-yw4l">Extension</td>
+     <td align="center"><b>Extension</b></td>
-     <td class="tg-yw4l">30-40 cycles<br></td>
+     <td align="center">30-40 cycles<br></td>
-     <td class="tg-yw4l">72°C</td>
+     <td align="center">72°C</td>
-     <td class="tg-yw4l">15-90s</td>
+     <td align="center">15-90s</td>
    </tr>
    <tr>
-     <td class="tg-yw4l">Final extension<br></td>
+     <td align="center"><b>Final extension</b></td>
-     <td class="tg-yw4l">1 cycle<br></td>
+     <td align="center">1 cycle<br></td>
-     <td class="tg-yw4l">72°C</td>
+     <td align="center">72°C</td>
-     <td class="tg-yw4l">5 min<br></td>
+     <td align="center">5 min<br></td>
    </tr>
 </table>
-<br> <br>
+<div style="text-indent:40px;">
-- Load probes on the agarose gel <br>
+. Load probes on the agarose gel. </div>
-- Store probes for short time on ice, for long at -20°C <br>
+<div style="text-indent:40px;">
+. Store probes for short time on ice, for long at -20°C. </div>
+<br>
-</div></div>
+<hr size="10" noshade></hr>
+<p style="font-size:15pt;"></sup><sup>[1]https://www.highqu.com/media/wysiwyg/ressources/manuals/PCM02_ALLin_Red_Taq_Mastermix_PI.pdf (accessed 31 October 2017) </p>
+</div></div></div>
@@ Line 297: / Line 421: @@
 <div class="spoiler">
-<input type="button" style="height: 50px; width: 50%; BACKGROUND-COLOR: #3399FF; font-size:25; color:black;" onclick="showSpoiler(this);" value="DNA Purification" />
+<input type="button" style="height: 50px; width: 50%; BACKGROUND-COLOR: rgb(61,67,80); font-size:25; color:white;" onclick="showSpoiler(this);" value="DNA Purification" />
 <div class="inner" style="display:none;">
-<br> <br>
+<br><br>
-<p align="center"><b>Depending on the PCR product</b> <p>
+<b>1. Aim </b> <br> <br>
+<div style="text-align: justify; margin-left:20px">
+. Purification of DNA
+after a gel electrophoresis</div><br> <br>
+<b> 2. Steps </b> <br> <br>
+<p align="center"><b>1. Depending on the PCR product</b> <p>
 <table>
    <tr>
@@ Line 319: / Line 448: @@
    </tr>
 </table>
-<br>
-<b>Binding of DNA </b>
+<b>2. Binding of DNA </b>
-<br> <br>
+<br><br><div style="text-align: justify; margin-left:40px">
-.  Insert SV Minicolumn into Collection Tube.
+.  Insert SV Minicolumn into Collection Tube.<br>
-<br>
+.  Transfer dissolved gel mixture or prepared PCR product to the Minicolumn assembly. Incubate at room temperature for 1 minute.
-.  Transfer dissolved gel mixture or prepared PCR product to the Minicolumn assembly. Incubate
-at room temperature for 1 minute.
 <br>
 .  Centrifuge at 16,000 ×g for 1 minute. Discard flowthrough and reinsert Minicolumn
-into Collection Tube.
+into Collection Tube.<br>
-<br>
+. Heat NE-buffer to 70 °C.</div>
-. Heat NE-buffer to 70 °C.
+<br>
-<br> <br>
+<b>3. Washing </b>
-<b>Washing </b>
+ <br><br><div style="text-align: justify; margin-left:40px">
-<br> <br>
+.  Add 700 μl Membrane Wash Solution (ethanol added). Centrifuge at 16,000 × g for 1 minute.
-.  Add 700 μl Membrane Wash Solution (ethanol added). Centrifuge at 16,000 × g for 1 minute.
+Discard flowthrough and reinsert Minicolumn into Collection Tube.<br>
-Discard flowthrough and reinsert Minicolumn into Collection Tube.
-<br>
+.  Repeat Step 4 with 500 μl Membrane Wash Solution. Centrifuge at 16,000 × g for 5 minutes.<br>
-.  Repeat Step 4 with 500 μl Membrane Wash Solution. Centrifuge at 16,000 × g for 5 minutes.
-<br>
+.   Empty the Collection Tube and recentrifuge the column assembly for 1 minute with the
-.   Empty the Collection Tube and recentrifuge the column assembly for 1 minute with the
+microcentrifuge lid open(or off to allow evaporation of any residual ethanol.</div>
-microcentrifuge lid open(or off to allow evaporation of any residual ethanol.
+<br>
-<br> <br>
+<b>4. Elution</b>
-<b>Elution</b>
+<br><br><div style="text-align: justify; margin-left:40px">
-<br> <br>
+.  Carefully transfer Minicolumn to a clean 1.5 ml microcentrifuge tube.<br>
-.  Carefully transfer Minicolumn to a clean 1.5 ml microcentrifuge tube.
-<br>
+.  Add 50 μl of Nuclease-Free Water to the Minicolumn. Incubate at room temperature for 1 minute. Centrifuge at 16,000 × g for 1 minute. <br>
-.  Add 50 μl of Nuclease-Free Water to the Minicolumn. Incubate at room temperature for 1 minute. Centrifuge at 16,000 × g for 1 minute.
-<br>
+.  Discard Minicolumn and measure the concentration.<br>
-.  Discard Minicolumn and measure the concentration.
-<br>
+. Store DNA at 4°C or –20°C.</div>
-. Store DNA at 4°C or –20°C.
 <br>
+<hr size="10" noshade></hr>
+<p style="font-size:12pt;"><sup>[1]</sup>https://www.promega.de/-/media/files/resources/protcards/wizard-sv-gel-and-pcr-clean-up-system-quick-protocol.pdf?la=de-de (accessed 31 October 2017)</p>
+</div></div></div>
-</div></div>
@@ Line 359: / Line 488: @@
 <div class="spoiler">
-<input type="button" style="height:50px; width:50%; BACKGROUND-COLOR: #3399FF; font-size:25; color:black;" onclick="showSpoiler(this);" value="Gel electrohoresis" />
+<input type="button" style="height: 50px; width: 50%; BACKGROUND-COLOR: rgb(61,67,80); font-size:25; color:white;" onclick="showSpoiler(this);" value="Gel electrohoresis" />
 <div class="inner" style="display:none;">
+<div align="justify">
+<br>
+<b>1. What is it ? </b><br><br>
+<div style="text-align: justify; margin-left:40px">
+. Standard lab procedure for separating DNA by size (e.g., length in base pairs) for visualization and purification. <br>
+. Uses an electrical field to move the negatively charged DNA through an agarose gel matrix  toward a positive electrode. <br>
-<b> What is it ? </b>
+. Shorter DNA fragments migrate through the gel more quickly than longer ones. </div>
-– standard lab procedure for separating DNA by size (e.g., length in base pairs) for visualization and purification <br>
-– uses an electrical field to move the negatively charged DNA through an agarose gel matrix  toward a positive electrode <br>
-- Shorter DNA fragments migrate through the gel more quickly than longer ones <br>
 <br>
-<b>Why are we doing it ? </b> <br>
+<b>2. Why are we doing it ? </b> <br><br>
-– to determine the approximate length of a DNA fragment by running it on an agarose gel alongside a DNA ladder (a collection of DNA fragments of known lengths) <br>
+<div style="text-align: justify; margin-left:40px">
+. to determine the approximate length of a DNA fragment by running it on an agarose gel alongside a DNA ladder (a collection of DNA fragments of known lengths). </div>
 <br>
-<b>Protocol : </b> <br>
+<div align="justify">
-- Pouring a Standard 1% Agarose Gel: <br>
+<b>3. Protocol  </b> <br><br>
-<div style="text-indent:10px;"> - Measure 1g agarose and and mix it with 100ml of TBE in a microwaveable flask. <br>
+<div style="text-indent:20px;">
-<div font-color="green"> Note:  Agarose gels are commonly used in concentrations of 0.7% to 2% depending on the size of bands needed to be separated -  Simply adjust the mass of agarose in a given volume to make gels of other agarose concentrations (e.g., 2 g of agarose in 100 mL of TAE will make a 2% gel). <br> </div> </div>
+. Pouring a Standard 1% Agarose Gel: </div>
-<br> <br>
+<div style="text-align: justify; margin-left:40px">
-- Microwave for 1-3 min until the agarose is completely dissolved (but do not overboil the solution, as some of the buffer will evaporate and thus alter the final percentage of agarose in the gel. Many people prefer to microwave in pulses, swirling the flask occasionally as the solution heats up.). <br>
+. Measure 1g agarose and and mix it with 100 ml of TBE in a microwaveable flask. <br>
-<div font-color="green"> Note: gloves and glasses ! Caution HOT! Be careful stirring, eruptive boiling can occur.<br>
+Note:  Agarose gels are commonly used in concentrations of 0.7 % to 2 % depending on the size of bands needed to be separated -  Simply adjust the mass of agarose in a given volume to make gels of other agarose concentrations (e.g., 2 g of agarose in 100 mL of TAE will make a 2 % gel).  <br>
+. Microwave for 1-3 min until the agarose is completely dissolved (but do not overboil the solution, as some of the buffer will evaporate and thus alter the final percentage of agarose in the gel. Many people prefer to microwave in pulses, swirling the flask occasionally as the solution heats up.). <br>
+Note: gloves and glasses ! Caution HOT! Be careful stirring, eruptive boiling can occur.<br>
 It is a good idea to microwave for 30-45 sec, stop and swirl, and then continue towards a boil. Keep an eye on it as the initial boil has a tendency to boil over. Placing saran wrap over the top of the flask can help with this, but is not necessary if you pay close attention. </div>
-<br> <br>
+<br>
-<b> Pouring of the gel </b> <br>
+<b> 4. Pouring of the gel </b> <br><br>
-- Let agarose solution cool down to about 50°C (about when you can comfortably keep your hand on the flask), about 5 mins. <br>
+<div style="text-align: justify; margin-left:40px">
-<div font-color="green"> Note:  or cool down in water bath about 30 min </div> <br>
+. Let agarose solution cool down to about 50 °C (about when you can comfortably keep your hand on the flask), about 5 mins. <br>
-- Add ethidium bromide (EtBr) to a final concentration of approximately 0.2-0.5 μg/mL (usually about 2-3 μl of lab stock solution per 100 mL gel). EtBr binds to the DNA and allows you to visualize the DNA under ultraviolet (UV) light. <br>
+Note:  Or cool down in water bath about 30 min. <br>
-<div font-color="green"> Note:  Caution EtBr is a known mutagen. Wear a lab coat, eye protection and gloves when working
+. Add ethidium bromide (EtBr) to a final concentration of approximately 0.2-0.5 μg/mL (usually about 2-3 μl of lab stock solution per 100 mL gel). EtBr binds to the DNA and allows you to visualize the DNA under ultraviolet (UV) light. <br>
+Note:  Caution EtBr is a known mutagen. Wear a lab coat, eye protection and gloves when working
 with this chemical. If you add EtBr to your gel, you will also want to add it to the running buffer when you run
-the gel. </div> <br>
+the gel. <br>
-- Pour the agarose into a gel tray with the well comb in place. <br>
+. Pour the agarose into a gel tray with the well comb in place. <br>
-<div font-color="green"> Note: Think about witch gel tray size you need. (a small one or a big one.)<br>
+ Note: Think about witch gel tray size you need (a small one or a big one).<br>
-  Pour slowly to avoid bubbles which will disrupt the gel. Any bubbles can be pushed away from the well comb or towards the sides/edges of the gel with a pipette trip. <br> </div>
+  Pour slowly to avoid bubbles which will disrupt the gel. Any bubbles can be pushed away from the well comb or towards the sides/edges of the gel with a pipette trip. <br>
-- Let the newly poured gel sit at room temperature for 20-30 mins, until it has completely solidified. <br>
+. Let the newly poured gel sit at room temperature for 20-30 mins, until it has completely solidified. <br>
-<div font-color="green"> if  you are in a hurry the gel can also be set more quickly if you place the gel tray at 4°C
+if  you are in a hurry the gel can also be set more quickly if you place the gel tray at 4 °C
-earlier so that it is already cold when the gel is poured into it. </div> <br> <br>
+earlier so that it is already cold when the gel is poured into it. </div> <br>
-<b> Loading Samples and Running an Agarose Gel: </b>
+<b>5. Loading Samples and Running an Agarose Gel </b>
-<br> <br>
+<br><br>
-- Add loading buffer to each of your digest samples. <br>
+<div style="text-align: justify; margin-left:40px">
+. Add loading buffer to each of your digest samples. <br>
 Note:
 Loading buffer serves two purposes: 1) it provides a visible dye that helps with gel
 loading and will also allows you to gauge how far the gel has run while you are running
 your gel; and 2) it contains a high percentage of glycerol, so it increases the density of your
-DNA sample causing it settle to the bottom of the gel well, instead of diffusing in the buffer.
+DNA sample causing it settle to the bottom of the gel well, instead of diffusing in the buffer.<br>
 .
-Once solidified, place the agarose gel into the gel box (electrophoresis unit).
+Once solidified, place the agarose gel into the gel box (electrophoresis unit).<br>
 .
-Fill gel box with 1xTAE (or TBE) until the gel is covered.
+Fill gel box with 1xTAE (or TBE) until the gel is covered.<br>
-.  Carefully load a molecular weight ladder into the first lane of the gel.
+.  Carefully load a molecular weight ladder into the first lane of the gel.<br>
 Note:
 When loading the sample in the well, maintain positive pressure on the sample to prevent
@@ Line 410: / Line 550: @@
 above the well. Very slowly and steadily, push the sample out and watch as the sample fills the well.
 After all of the sample is unloaded, push the pipettor to the second stop and carefully raising the
-pipette straight out of the buffer.
+pipette straight out of the buffer.<br>
-.  Carefully load your samples into the additional wells of the gel.
+.  Carefully load your samples into the additional wells of the gel.<br>
-.  Run the gel at 80-150 V until the dye line is approximately 75-80% of the way down the gel.
+.  Run the gel at 80-150 V until the dye line is approximately 75-80 % of the way down the gel.<br>
 Note:
   Black is negative, red is positive. (The DNA is negatively charged and will run towards the
 positive electrode.)
-Always Run to Red.
+Always Run to Red. <br>
 Note:
-  A typical run time is about 1-1.5 hours, depending on the gel concentration and voltage.
+  A typical run time is about 1-1.5 hours, depending on the gel concentration and voltage.<br>
 .   Turn OFF power, disconnect the electrodes from the power source, and then carefully   remove
-the gel from the gel box.
+the gel from the gel box.<br>
-. Using any device that has UV light, visualize your DNA fragments.
+. Using any device that has UV light, visualize your DNA fragments.<br>
-Note:
-Fabian or Lena will give you a short introduction on how to work with UV light !!!
 Note:
 When using UV light, protect your skin by wearing safety goggles or a face shield, gloves
-and a lab coat.
+and a lab coat.<br>
 Note:
 If you will be purifying the DNA for later use, use long-wavelength UV and expose for as
-little time as possible to minimize damage to the DNA.
+little time as possible to minimize damage to the DNA.<br>
 Note:
-The fragments of DNA are usually referred to as ‘bands’ due to their appearance on the gel.
+The fragments of DNA are usually referred to as ‘bands’ due to their appearance on the gel.<br></div><br>
-Analyzing Your Gel:
+<b>6. Analyzing Your Gel</b><br><br>
+<div style="text-align: justify; margin-left:20px">
 Using the DNA ladder in the first lane as a guide (the manufacturer's instruction will tell you the
 size of each band), you can interpret the bands that you get in your sample lanes to determine if the
@@ Line 438: / Line 578: @@
 digests and how to interpret them please see the
 Diagnostic Digest
-page.
+page. </div><br>
-Purifying DNA from Your Gel:
+<b>7. Purifying DNA from Your Gel</b><br><br>
+<div style="text-align: justify; margin-left:20px">
 If you are conducting certain procedures, such as molecular cloning, you will need to purify the
 DNA away from the agarose gel. For instructions on how to do this, visit the
 Gel Purification
-page
+page.<br></div><br> <br><br>
+<hr size="10" noshade></hr>
-  </div></div>
+<p style="font-size:15pt;"><sup>[1]
+http://www.addgene.org/protocols/gel-electrophoresis/ (accessed 31 October 2017)</p>
+  </div></div></div>
+<div class="spoiler">
+<input type="button" style="height: 50px; width: 50%; BACKGROUND-COLOR: rgb(61,67,80); font-size:25; color:white;" onclick="showSpoiler(this);" value="Ligation" />
+<div class="inner" style="display:none;">
+<div align="justify">
+<br> <br>
+<b>1. Aim </b> <br> <br>
+<div style="text-align: justify; margin-left:20px">
+. Enzymatically linkage of two DNA/RNA segments. <br><br>
+</div>
+<b> 2. Steps </b> <br> <br>
+<div style="text-align: justify; margin-left:20px">
+. Set up the following reaction in a microcentrifuge tube on ice.<br></div><br> <br>
+<table>
+  <tr>
+    <th  width="50%" align="center"><b>component</b></th>
+    <th  width="50%" align="center"><b>volume</b></th>
+  </tr>
+  <tr>
+    <td align="center">T4 DNA Ligase Buffer (10 x)</td>
+    <td align="center">2 µl</td>
+  </tr>
+  <tr>
+    <td align="center">10x buffer</td>
+    <td align="center">1 µl</td>
+  </tr>
+  <tr>
+    <td align="center">T4 DNA Ligase</td>
+    <td align="center">1 µl</td>
+  </tr>
+  <tr>
+    <td align="center"> Vector DNA </td>
+    <td align="center"></td>
+  </tr>
+  <tr>
+    <td align="center">Insert DNA</td>
+    <td align="center"></td>
+  </tr>
+  <tr>
+    <td align="center">Nuclease-free water </td>
+    <td align="center"> to 20 µl</td>
+  </tr>
+</table>
+<div style="text-align: justify; margin-left:60px">
+. Calculation of the DNA
+<br>
+kb (smaller DNA)/ kb (larger DNA)&sdot;mass (Vector DNA)&sdot;relation (Insert DNA)
+<br> <br>
+Example calculation </div>
+<div style="text-align: justify; margin-left:80px">
+:3 vector to insert <br>
+mass Vector DNA: 100 ng <br>
+Vector DNA: 10 kb <br>
+Insert DNA: 3 kb<br><br>
+kb/ 10 kb&sdot;100 ng&sdot;3 = 90 ng</div>
+<div style="text-align: justify; margin-left:60px">
+. T4 DNA Ligase should be added last.<br>
+. Use nebiocalculator.neb.com/#!/ to calculate molar ratios. <br>
+. The T4 DNA Ligase Buffer should be thawed and resuspended at room temperature. </div>
+<div style="text-align: justify; margin-left:20px">
+. Gently mix the reaction by pipetting up and down and microfuge briefly.<br>
+. Incubation </div>
+<div style="text-align: justify; margin-left:60px">
+. Cohesive (sticky) ends. </div>
+<div style="text-align: justify; margin-left:80px"> 1. 16 °C overnight or room temperature for 10 minutes. </div>
+<div style="text-align: justify; margin-left:60px">
+. Blunt ends or single base overhangs. </div>
+<div style="text-align: justify; margin-left:80px">
+. 16°C overnight or room temperature for 2 hours (alternatively, high concentration T4 DNA Ligase can be used in a 10 minute ligation). </div>
+<div style="text-align: justify; margin-left:20px">
+.Heat inactivate at 65°C for 10 minutes. <br>
+. Chill on ice and transform 1-5 μl of the reaction into 50 μl competent cells.</div>
+<br> <br>
+<hr size="10" noshade></hr>
+<p style="font-size:12pt;"><sup>[1]</sup>
+https://www.neb.com/protocols/1/01/01/dna-ligation-with-t4-dna-ligase-m0202 (accessed 31 October 2017)</p>
+</div></div></div></div>
@@ Line 452: / Line 678: @@
 <div class="spoiler">
-<input type="button" style="height:50px; width:50%; BACKGROUND-COLOR: #3399FF; font-size:25; color:black;" onclick="showSpoiler(this);" value="PCR" />
+<input type="button" style="height: 50px; width: 50%; BACKGROUND-COLOR: rgb(61,67,80); font-size:25; color:white;" onclick="showSpoiler(this);" value="Miniprep"/>
 <div class="inner" style="display:none;">
-What is the PCR ?
+<br> <br>
-Method to make multiple copies of a
+<b> 1. Aim </b><br> <br>
-the specific  DNA-sequence
+<div style="text-indent:20px;">
-Protocol for PCR with Q5 High- Fidelity 2x Master Mix
+Isolation of DNA as a plasmid</div>
-Please note that protocols with
+<br> <br>
-Q5 High-Fidelity DNA Polymerase may differ from protocols
+<b>2.Production of cleared lysate</b>
-with other polymerases. Conditions recommended below should be used for optimal
+<br><br>
-performance.
+<div style="text-indent:20px;">
-Reaction Setup:
+. Isolation of the bacteria </div>
-–
+<div style="text-align: justify; margin-left:40px">
-assemble all reaction components on ice, work on ice while assembling
+.  Harvest 1–5 ml (high-copy-number plasmid) or 10 ml (low-copy-number plasmid)
-–
+of bacterial culture .
-preheat the thermocycler to the denaturation temperature( 98 °C)
+<br>
-–
+. Centrifugation for 5 minutes at 10,000 xg in a tabletop centrifuge.
-prior to use all components should be mixed
+<br>
-–
+. Pour off the supernatant.
-work quickly when transferring the reactions to a thermocycler
+<br>
-.
+. Reinsert again bacterial culture to the pellet and repeat step 2 and 3.
-on ice
+<br>
-  Assemble all components for the reaction :
+. Blot the inverted tube on a paper towel to remove excess media.
-Component
+<br> </div>
-μl Reaction
+<div style="text-indent:20px;">
-μl Reaction
+.
-Final Concentration
+Resuspension of the cells </div>
-Q5
+<div style="text-align: justify; margin-left:40px">
-High-Fidelity 2X Master Mix
-.5 μl
-μl
-X
-μM Forward Primer
-.25 μl
-.5 μl
-.5 μM
-μM Reverse Primer
-.25 μl
-.5 μl
-.5 μM
-Template DNA
-variable
-variable
-< 1,000 ng
-Nuclease-Free Water
-to 25 μl
-to 50 μl
-Notes: Two Primers have to be diluted 1:10 !
-Notes: Gently mix the reaction. Collect all liquid to the bottom of the tube by a quick spin if
-necessary. Overlay the sample with mineral oil if using a PCR machine without a heated lid.
-. Transfer PCR tubes to a PCR machine and begin thermocycling.
- Steps
-of
- PCR:
-.Denaturation : double- stranded template DNA is heated to separate it into two single stands
-. Annealing    :  temperature is lowered to enable the DNA primers to attach to the template DNA
-. Extending    : temperature is raised and the new strand of DNA is made by the  polymerases
-Thermocycling Conditions for a Routine PCR:
-STEP
-TEMP
-TIME
-Initial Denaturation
-°C
-seconds
-–35 Cycles
-°C
-–10 seconds
-*50–72°C
-–30
-seconds
-°C
-–30
-seconds
-/kb
-Final Extension
-°C
-minutes
-Hold
-–10°C
-hold is not
-necessary
 .
-Template:
+Add 250 μl of Cell Resuspension Solution.
-Use of high quality, purified DNA templates greatly enhances the success of PCR.
+<br>
-Recommended amounts of DNA template for a 50 μl reaction are as follows:
-DNA
-AMOUNT
-DNA Genomic
-ng–1 μg
-Plasmid or Viral
-pg–1 ng
 .
-Primers:
+Completely resuspend the cell pellet by vortexing or pipetting.
-Oligonucleotide primers are generally 20–40 nucleotides in length and ideally have a GC
+<br>
-content of 40–60%. Computer programs such as
-Primer3
- can be used to design or analyze
-primers. The best results are typically seen when using each primer at a final concentration
-of 0.5 μM in the reaction.
 .
-Mg
+It is essential to thoroughly resuspend the cells.
-++
+<br></div>
- and additives:
+<div style="text-indent:20px;">
-The
+.
-Q5 High-Fidelity Master Mix contains 2.0
+Lysing </div>
-mM Mg
+<div style="text-align: justify; margin-left:40px">
-++
+.
- when used at a 1X concentration.
+Add 250 μl of Cell Lysis Solution.
-This is optimal for most PCR products generated with this master mix.
+<br>
+.
+Mix by inverting the tube 4 times - do not vortex.
+<br>
+.
+Incubate until the cell suspension clears (clear ≠ colorlessly) (approximately 1–5 minutes).
+<br></div>
+<div style="text-indent:20px;">
 .
-Deoxynucleotides:
+Splitting proteins </div>
-The final concentration of dNTPs is 200 μM of each deoxynucleotide in the 1X
+<div style="text-align: justify; margin-left:40px">
-Q5 High-
+.
-Fidelity Master Mix.
+Add 10 μl of Alkaline Protease Solution.
-Q5 High-Fidelity DNA Polymerase cannot incorporate dUTP and is not
+<br>
-recommended for use with uracil-containing primers or templates.
+.
+Mix by inverting the tube 4 times - do not vortex.
+<br>
+.
+Incubate for 5 minutes at room temperature.
+<br></div>
+<div style="text-indent:20px;">
 .
-Q5
+Neutralization </div>
-High-Fidelity DNA Polymerase concentration:
+<div style="text-align: justify; margin-left:40px">
-The concentration of
+.
-Q5 High-Fidelity DNA Polymerase in the
+Add 350 μl of Neutralization Solution.
-Q5 High-Fidelity 2X Master
+<br>
-Mix has been optimized for best results under a wide range of conditions.
+.
+Immediately mix by inverting the tube 4 times - do not vortex.
+<br></div>
+<div style="text-indent:20px;">
 .
-Denaturation:
+Isolation of the plasmids </div>
-An initial denaturation of 30 seconds at 98°C is sufficient for most amplicons from pure
-DNA templates. Longer denaturation times can be used (up to 3 minutes) for templates that
-require it.
-During thermocycling, the denaturation step should be kept to a minimum. Typically, a 5–10
-second denaturation at 98°C is recommended for most templates.
-.
-Annealing:
-Optimal annealing temperatures for
-Q5 High-Fidelity DNA Polymerase tend to be higher
-than for other PCR polymerases. The
-NEB T
-m
- Calculator
- should be used to determine the
-annealing temperature when using this enzyme. Typically use a 10–30 second annealing step
-at 3°C above the T
-m
- of the lower T
-m
- primer. A temperature gradient can also be used to
-optimize the annealing temperature for each primer pair.
-For high T
-m
- primer pairs, two-step cycling without a separate annealing step can be used
-(see note 10).
-.
-Extension:
-The recommended extension temperature is 72°C. Extension times are generally 20–30
-seconds per kb for complex, genomic samples, but can be reduced to 10 seconds per kb for
-simple templates (plasmid,
-E. coli
-, etc.) or complex templates < 1 kb. Extension time can be
-increased to 40 seconds per kb for cDNA or long, complex templates, if necessary.
-A final extension of 2 minutes at 72°C is recommended.
-.
-Cycle number:
-Generally, 25–35 cycles yield sufficient product.
-For genomic amplicons, 30-35 cycles are
-recommended.
-.
--step PCR:
-When primers with annealing temperatures ≥
-°C are used, a 2-step thermocycling protocol
-(combining annealing and extension into one step) is possible.
-.
-Amplification of long products:
-When amplifying products > 6 kb, it is often helpful to increase the extension time to 40–50
-seconds/kb.
-.
-PCR product:
-The PCR products generated using
-Q5 High-Fidelity
-X
-Master Mix
-have blunt ends. If
-cloning is the next step, then blunt-end cloning is recommended. If T/A-cloning is preferred,
-the DNA should be purified prior to A-addition, as
-Q5 High-Fidelity DNA Polymerase will
-degrade any overhangs generated.
-Addition of an untemplated -dA can be done with
-Taq
-DNA Polymerase (
-NEB #M0267
- ) or
-Klenow exo
-–
- (
-NEB #M0212
- )
-</div></div>
+<div style="text-align: justify; margin-left:40px">
+.
+Centrifuge the bacterial lysate at maximum speed (around 14,000 ×g) in a microcentrifuge for 10 minutes at room temperature.
+<br> <br>
+<br></div>
+<b>3. Isolation of the plasmid DNA </b>
+<br><br>
+<div style="text-align: justify; margin-left:20px">
+. Transfer the cleared lysate (approximately 850 μl, Section 3.B, Step 6) to the
+prepared Spin Column by decanting. Avoid disturbing or transferring any of the
+white precipitate with the supernatant. </div>
+<div style="text-align: justify; margin-left:40px">
+. If the white precipitate is accidentally transferred to the Spin Column, pour
+the Spin Column contents back into a sterile 1.5ml microcentrifuge tube
+and centrifuge for another 5–10 minutes at maximum speed. Transfer the
+resulting supernatant into the same Spin Column that was used initially for
+this sample. The Spin Column can be reused but only for this sample.</div>
+<div style="text-align: justify; margin-left:20px">
+. Centrifuge the supernatant at maximum speed in a microcentrifuge for 1 minute at
+room temperature. Remove the Spin Column from the tube and discard the
+flowthrough from the Collection Tube. Reinsert the Spin Column into the Collection
+Tube. </div>
+<div style="text-align: justify; margin-left:20px">
+. Wash the plasmid DNA. </div>
+<div style="text-align: justify; margin-left:40px">
+. Add 750 μl of Column Wash Solution.
+<br>
+. Centrifuge at maximum speed in a microcentrifuge for 1 minute at room
+temperature.
+<br>
+. Remove the Spin Column from the tube and discard the flowthrough.
+<br>
+. Reinsert the Spin Column into the Collection Tube.
+<br></div>
+<div style="text-align: justify; margin-left:20px">
+. Wash again the plasmid DNA. </div>
+<div style="text-align: justify; margin-left:40px">
+. Add 250 μl of Column Wash Solution.
+<br>
+. Centrifuge at maximum speed in a microcentrifuge for 2 minutes at room
+temperature.
+<br>
+. If the Spin Column has Column Wash Solution associated with it,
+centrifuge again for 1 minute at maximum speed.
+<br>
+. Transfer the Spin Column to a new, sterile 1.5ml microcentrifuge tube, being
+careful not to transfer any of the Column Wash Solution with the Spin Column.
+<br></div>
+<div style="text-indent:20px;">
+. Elute the plasmid DNA </div>
+<div style="text-align: justify; margin-left:40px">
+. Add 50 μl of Nuclease-Free Water to the Spin Column, wait 5 minutes
+<br>
+. Centrifuge at maximum speed for 1 minute at room temperature in a
+microcentrifuge.
+<br></div>
+<div style="text-align: justify; margin-left:20px">
+. After eluting the DNA, remove the assembly from the 1.5ml microcentrifuge tube
+and discard the Spin Column.</div>
+<br>
+<hr size="10" noshade></hr>
+<p style="font-size:12pt;"><sup>[1]</sup>https://www.promega.de/-/media/files/resources/protocols/technical-bulletins/0/wizard-plus-sv-minipreps-dna-purification-system-protocol.pdf (accessed 31 October 2017)</p>
+ </div></div>
 <div class="spoiler">
-<input type="button" style="height:50px; width:50%; BACKGROUND-COLOR: 	#3399FF; font-size:25; color:black;" onclick="showSpoiler(this);" value="Restriction" />
+<input type="button" style="height: 50px; width: 50%; BACKGROUND-COLOR: rgb(61,67,80); font-size:25; color:white;" onclick="showSpoiler(this);" value="PCR" />
-<div class="inner" style="display:none;"> Protocol is following! </div></div>
+<div class="inner" style="display:none;">
+<br> <br>
+<b>1. What is the PCR ? </b><br><br>
+<b>p</b>olymerase <b>c</b>hain <b>r</b>eaction<br>
+Method to make multiple copies of a
+the specific  DNA-sequence <br><br>
+<b>2. Reaction Setup</b><br><br>
+<div style="text-align: justify; margin-left:40px">
+. Assemble all reaction components on ice, work on ice while assembling. <br>
+. Preheat the thermocycler to the denaturation temperature (98 °C). <br>
-<div class="spoiler">
+. Prior to use all components should be mixed. <br>
-<input type="button"  style="height:50px; width:50%; BACKGROUND-COLOR: 	#3399FF; font-size:25; color:black;" onclick="showSpoiler(this);" value="Salkowski Assey" />
-<div class="inner" style="display:none;">
-Salkowski Assay for colony screening
+. Work quickly when transferring the reactions to a thermocycler. </div><br><br>
-Large qualitative screening of IAA-producing colonies at the same time to see if our constructs are still functional in our E.coli/yeasts
+<b>3. Steps </b><br><br>
-Helps us pic kthe right colonies for colony-PCR and GC-MS measurements
+<div style="text-align: justify; margin-left:40px">
-Reagent: 2% 0.5M FeCl3 in 35% perchloric acid
+. Assemble all components on ice for the reaction :<br><br>
-Perchloric acid is highly corrosive and dangerous!! Always uses protective gear and work under a fume hood!
+<table>
-Reagent will always be mixed together on the spot, FeCl3 stock solution is finished, acid will be taken from the chemicals sheld from the AG plant physiology (has been negotiated)
+  <tr>
-What happens?
+    <th class="tg-yw4l" align="center"><b>Component</b></th>
-Reagent reacts to IAA (and other indolic compounds) to make several colored products
+    <th class="tg-yw4l" align="center"><b>25 μl Reaction</b></th>
-IAA will be seen as bright red (other compounds brown or yellowish)
+    <th class="tg-yw4l" align="center"><b>
-Assay conditions
+μl Reaction</b></th>
-Plates were inoculated in a grid pattern and overlaid with an 82mm-diameter disk of Nitrocellulose membranes
+    <th class="tg-yw4l" align="center"><b>Final Concentration</b></th>
-Plates are overlaid with Nitrocellulose immediately after inoculation with toothpicks
+  </tr>
-After normal incubation (i.e. overnight) time, the membrane was removed and soaked in reagent (or reagent-saturated [2.5 mL] filter paper, here “Whatman grade 2” had best results))
+  <tr>
-After 30 - 60 minutes, coloring reaction is finished and fading began
+    <td class="tg-yw4l" align="center"><b>Q5
-Best results with colony sizes between 0.5 to 2 mm
+High-Fidelity 2X Master Mix</b></td>
-Addition of Tryptophan greatly enhances color reaction but does not interfere with distinguishing IAA positive and negative colonies (yellow background and strong red to pink positives)
+    <td class="tg-yw4l" align="center">12.5 μl</td>
-Other indolic compounds (i.e. indolepyruvic acid) are distinguishable by a more yellow-brownish color
+    <td class="tg-yw4l" align="center">25 μl</td>
+    <td class="tg-yw4l" align="center">1X</td>
+  </tr>
+  <tr>
+    <td class="tg-yw4l" align="center"><b>10 μM Forward Primer</b></td>
+    <td class="tg-yw4l" align="center">1.25 μl</td>
+    <td class="tg-yw4l" align="center">
+.5 μl</td>
+    <td class="tg-yw4l"align="center">0.5 μM</td>
+  </tr>
+  <tr>
+    <td class="tg-yw4l"align="center">
+<b>10 μM Reverse Primer</b></td>
+    <td class="tg-yw4l"align="center">1.25 μl</td>
+    <td class="tg-yw4l"align="center">2.5 μl</td>
+    <td class="tg-yw4l"align="center">0.5 μM</td>
+  </tr>
+  <tr>
+    <td class="tg-yw4l"align="center">
+<b>Template DNA</b></td>
+    <td class="tg-yw4l"align="center">variable</td>
+    <td class="tg-yw4l"align="center">variable</td>
+    <td class="tg-yw4l"align="center">< 1,000 ng</td>
+  </tr>
+  <tr>
+    <td class="tg-yw4l"align="center">
+<b>Nuclease-Free Water</b></td>
+    <td class="tg-yw4l"align="center">to 25 μl</td>
+    <td class="tg-yw4l"align="center">to 50 μl</td>
+    <td class="tg-yw4l"align="center"></td>
+  </tr>
+</table>
- </div></div>
+<br>
+Notes: Two Primers have to be diluted 1:10 ! <br>
+Notes: Gently mix the reaction. Collect all liquid to the bottom of the tube by a quick spin if
+necessary. Overlay the sample with mineral oil if using a PCR machine without a heated lid.<br>
+. Transfer PCR tubes to a PCR machine and begin thermocycling.</div>
+<br> <br>
+<b>4. Steps
+of
+ PCR</b><br><br>
+<div style="text-align: justify; margin-left:40px">
+.Denaturation : double- stranded template DNA is heated to separate it into two single stands. <br>
+. Annealing    :  temperature is lowered to enable the DNA primers to attach to the template DNA. <br>
+. Extending    : temperature is raised and the new strand of DNA is made by the  polymerases. <br>
+Thermocycling Conditions for a Routine PCR: </div><br>
+<table>
+    <tr>
+      <th align="center"><b>Step</b></th>
+      <th align="center"><b>Temperature</b></th>
+      <th align="center"><b>Time</b></th>
+    </tr>
+    <tr>
+      <td align="center"><b>Initial Denaturation</b></td>
+      <td align="center">98°C</td>
+      <td align="center">30 seconds </td>
+    </tr>
+    <tr>
+      <td align="center"><b></b></td>
+      <td align="center">98°C</td>
+      <td align="center">5–10 seconds</td>
+    </tr>
+ <tr>
+      <td align="center"><b>25–35 Cycles</b></td>
+      <td align="center">*50–72°C</td>
+      <td align="center">10–30 seconds</td>
+    </tr>
+ <tr>
+      <td align="center"><b></b></td>
+      <td align="center">72°C</td>
+      <td align="center">20–30 seconds/kb</td>
+    </tr>
+ <tr>
+      <td align="center"><b>Final Extension</b></td>
+      <td align="center">72°C</td>
+      <td align="center">2 minutes</td>
+    </tr>
+ <tr>
+      <td align="center"><b>Hold</b></td>
+      <td align="center">4-10°C</td>
+      <td align="center"></td>
+    </tr>
+</table>
+<br> <br>
+Please note that protocols with
+Q5 High-Fidelity DNA Polymerase may differ from protocols
+with other polymerases. Conditions recommended below should be used for optimal
+performance.<br><br>
+<hr size="10" noshade></hr>
+<p style="font-size:12pt;"><sup>[1]</sup>
+https://www.neb.com/protocols/2012/12/07/protocol-for-q5-high-fidelity-2x-master-mix-m0492 (accessed 31 October 2017)</p>
+</div></div>
@@ Line 694: / Line 968: @@
 <div class="spoiler">
-<input type="button" style="height: 50px; width: 50%; BACKGROUND-COLOR: #3399FF; font-size:25; color:black;" onclick="showSpoiler(this);" value="Transformation (E.Coli)" />
+<input type="button" style="height: 50px; width: 50%; BACKGROUND-COLOR: rgb(61,67,80); font-size:25; color:white;" onclick="showSpoiler(this);" value="Restriction" />
 <div class="inner" style="display:none;">
+<br>
+<b> 1. Aim </b><br><br>
+<div style="text-align: justify; margin-left:20px">
+A restriction digest is the division of DNA in a  specific area by the help of restriction enzymes. The aim afterwards could be to analyze and characterize (restriction maps) the DNA, to compare it to others or to clone it into e.g. a vector. Previous to the restriction, the DNA has to be isolated (see protocol miniprep).</div>
+<br>
+<b> 2. Test digest - What for?</b><br><br>
+<div style="text-align: justify; margin-left:20px">
+In a test digest the cleavage products are analyzed to verify that the used DNA has e.g. taken in a specific fragment. Only few of the DNA is digested, because the uncut DNA will be used in further steps. The whole DNA strand is often too long to analyze, therefore:</div><br>
+<ul style="text-align: justify; margin-left:40px;list-style-type:disc" float:right;>
+   <li>targeted cuts between specific base sequences </li>
+   <li>cleavage fragments small enough to run on gel </li>
+   <li>segregation and analysis by gel electrophoresis </li>
+</ul>
+<div style="text-align: justify; margin-left:20px">
+→ always with a control: uncut plasmid (see protocol gel electrophoresis)
+In a preparative digest normally 1 U enzyme digests 1 µg DNA in one hour. </div><br>
+<b>3. Preparative digest - What for? </b><br><br>
+<div style="text-align: justify; margin-left:20px">
+In a preparative digest the entire available DNA is digested, because the cleavage products are used in further steps. The cut DNA can be extracted from a gel.
+It is important that as much DNA as possible is digested. Therefore 0,2 - 0,4 µl enzyme per µg DNA are applied and the reaction should run for ~2h.</div>
+<br>
+<b> 4. Procedure</b><br><br>
+<div style="text-align: justify; margin-left:20px">
+exemplary pipetting scheme:</div>
+<br>
+<table>
+  <tr>
+    <th  width="50%" align="center"><b>component</b></th>
+    <th  width="50%" align="center"><b>volume</b></th>
+  </tr>
+  <tr>
+    <td align="center">DNA (typically 200 - 500 ng)</td>
+    <td align="center">1 µl </td>
+  </tr>
+  <tr>
+    <td align="center">10x buffer</td>
+    <td align="center">1 µl</td>
+  </tr>
+  <tr>
+    <td align="center">H<sub>2</sub>O</td>
+    <td align="center">7,6 µl</td>
+  </tr>
+  <tr>
+    <td align="center"> enzyme 1</td>
+    <td align="center">0,1 µl</td>
+  </tr>
+  <tr>
+    <td align="center">enzyme 2</td>
+    <td align="center">0,1 µl </td>
+  </tr>
+  <tr>
+    <td align="center">final volume </td>
+    <td align="center">10 µl</td>
+  </tr>
+</table>
+<br>
+<br>
+<div style="text-align: justify; margin-left:20px">
+If more than one reaction is done, it is convenient to prepare a master mix containing everything except for the DNA (!). This saves time and tips and keeps you from having to pipet very small volumes (e.g. 0,1 µl).<br>
-Tranformation Protocol
+It is also possible to use more than two or just one restriction enzyme per reaction. They just need to have the same buffer preferences. Volumes have to be adjusted to the number of enzymes.</div>
-What is it?
-Transmission of genetic information into competent cells (or plants, algas, mushrooms) (the target organismn)
-Übertragung von genetischer Information durch Aufnahme freier DNA in kompetente Zellen oder auch in Pilzen/Algen/Pflanzen
-DNA in Zielorganismen einzuschleusen
-Why we are doing it?
-Transmission of genetic information into competent cells
-What you need:
-*1.5 mL tube, pipette (50µL), pipette(1000µL), plate
-*Ice, SOC Medium, competent cells
-How we are doing it?
-. Prechil 1.5 ml tube on ice
-. Thaw a tube competent E. coli cells on ice for 10 minutes.
+<hr size="10" noshade></hr>
-.1.  mix gently
+<p style="font-size:12pt;"><sup>[1]</sup> http://www.log2embl.de/sites/default/files/Labor-Protokoll-Restriktionsverdau.pdf (accessed 30 October 2017) </p>
-.2. Pipette 50 µl of the cells  into the 1.5ml tube
-    (Temperature over 0°C decrease the efficiency of the transformation!)
+<p style="font-size:12pt;"><sup>[2]</sup>
+https://www.uni-hohenheim.de/fileadmin/einrichtungen/pflanzenphysiologie/Protokolle/V.Klonierung/restriktionsverdau_de.pdf (accessed 30 October 2017)</p>
-. Add 1-5 µl (containing 1 pg-100 ng of plasmid) DNA to the cell mixture.
-(as soon as as the last bit of ice in the tube is disappeared!)
+<p style="font-size:12pt;"><sup>[3]</sup>
+http://www.biochemie.uni-jena.de/files/Praktikum/plasmid%20dna%20+%20restrictionsverdau.pdf (accessed 30 October 2017)</p>
-. Flick the tube 4-5 times to mix cells and DNA.
-(No vortexing!)
-. Place the mixture on ice for 30 minutes
+</div></div>
-(without mixing!)
-(2-fold loss in transformation efficiency for every 10 minutes this step is shortened!)
-. Heat shock at exactly 42°C for exactly 30 seconds.
-(without mixing!)
-(temperature and timing specific to transformation volume and vessel)
-. Place on ice for 5 minutes.
-(without mixing!)
+<div class="spoiler">
+<input type="button" style="height: 50px; width: 50%; BACKGROUND-COLOR: rgb(61,67,80); font-size:25; color:white;" onclick="showSpoiler(this);" value="Salkowski Assay" />
+<div class="inner" style="display:none;">
+<br> <br>
+<b>1. Aim </b>
+<br><br>
+<div style="text-align: justify; margin-left:40px">
+. Large qualitative screening of IAA-producing colonies at the same time to see if our constructs are still functional in our E.coli/yeasts. <br>
+. Helps us to pick the right colonies for colony-PCR and GC-MS measurements. </div>
+<br>
+<b>2. Safty </b><br> <br>
+<div style="text-align: justify; margin-left:40px">
+. Reagent: 2 % 0.5M FeCl3 in 35 % perchloric acid </div>
+<div style="text-align: justify; margin-left:60px">
+. Perchloric acid is highly corrosive and dangerous!! Always uses protective gear and work under a fume hood! <br>
-. Pipette 950 µl of room temperature SOC into the mixture.
+. Reagent will always be mixed together on the spot, FeCl<sub>3</sub> stock solution is finished, acid will be taken from the chemicals sheld from the AG plant physiology (has been negotiated).</div>
+<br>
-. Place at 37°C for 60 minutes and shake vigorously (800 rpm in thermo mix block)
+<b>3. What happens? </b><br> <br>
+<div style="text-align: justify; margin-left:40px">
+. Reagent reacts to IAA (and other indolic compounds) to make several colored products. <br>
-(2-fold loss in transformation efficiency for every 15 minutes this step is shortened)
+. IAA will be seen as bright red (other compounds brown or yellowish).</div>
-(SOC gives 2-fold higher transformation efficiency than LB medium)
+<br>
-(incubation with shaking or rotating the tube gives 2-fold higher transformation efficiency than without)
+<b>4. Assay conditions </b> <br> <br>
+<div style="text-align: justify; margin-left:40px">
+. Plates were inoculated in a grid pattern and overlaid with an 82 mm-diameter disk of Nitrocellulose membranes.<br>
+. Plates are overlaid with Nitrocellulose immediately after inoculation with toothpicks.
+After normal incubation (i.e. overnight) time, the membrane was removed and soaked in reagent (or reagent-saturated [2.5 mL] filter paper, here “Whatman grade 2” had best results), in glass chamber ( danger symbol and written information).<br>
-. Warm selection plates to 37°C
+. After 30 - 60 minutes, coloring reaction is finished and fading began. <br>
-(plates can be used warm or cold, wet or dry…efficiency is nearly the same… warm plates easier to spread and allow most rapid colony formation)
+. Best results with colony sizes between 0.5 to 2 mm. <br>
-. Mix the cells thoroughly by flicking the tube and inverting
+. Addition of Tryptophan greatly enhances color reaction but does not interfere with distinguishing IAA positive and negative colonies (yellow background and strong red to pink positives).<br>
-. Spread 50-200 µl onto a selection plate and incubate overnight at 37°C.
+. Other indolic compounds (i.e. indolepyruvic acid) are distinguishable by a more yellow-brownish color. </div>
+<br>
+<b> 5. Afterwards </b><br> <br>
+<div style="text-align: justify; margin-left:40px">
+. Neutralize the acid with NaOH and use a pH-test strip. <br>
-(Alternative: incubate at 30°C for 24-36 hours or 25°C for 48 hours.)
+. Throw away liquid and solid waste separately. </div>
-(For low efficiency cloning reactions: spin down the whole transformation mixture, remove nearly the complete supernatant (approx. 900 ul), resuspend cells in remaining liquid and plate completely)
+ <br><br>
- </div></div>
+<hr size="10" noshade></hr>
+<p style="font-size:12pt;"><sup>[1]</sup>Bric JM, Bostock RM, Silverstone SE. Rapid In Situ Assay for Indoleacetic Acid Production by Bacteria Immobilized on a Nitrocellulose Membrane. Applied and Environmental Microbiology. 1991;57(2):535-538. (accessed 31 October 2017)</p>
+ </div></div>
@@ Line 766: / Line 1,109: @@
 <div class="spoiler">
-<input type="button"  style="height:50px; width:50%; BACKGROUND-COLOR: 	#3399FF; font-size:25; color:black;"  onclick="showSpoiler(this);" value="Transformation (yeast)" />
+<input type="button" style="height: 50px; width: 50%; BACKGROUND-COLOR: rgb(61,67,80); font-size:25; color:white;" onclick="showSpoiler(this);" value="SLiCE" />
 <div class="inner" style="display:none;">
+<br> <br>
+<b>1. Aim </b><br> <br>
+<div style="text-align: justify; margin-left:20px;">
+. Cloning method that uses bacterial cell extracts. <br>
+. Assembly  of  multiple  DNA  fragments  into  a  recombinant  DNA  molecule → in  a single  in  vitroreaction.<br>
+. General principle: recombining short end homologies (15-52 bp.)<br>
+. Homologous  ends  can  be  flanked  by  heterologous  sequences  (e.g.  for  inducing  a  linker sequence).</div><br> <br>
+<b>2. Steps </b><br><br>
+<div style="text-align: justify; margin-left:20px">
+No steps have
+to be done at the clean bench, working at room temperature.</div>
+<div style="text-align: justify; margin-left:20px">
+. Prepare 10X SLiCE Buffer in a 1.5 mL tube : </div>
+<div style="text-align: justify; margin-left:40px">
+μL 1 M Tris-HCl pH 7.5 <br>
++     50 μL 2 M MgCl<sub>2</sub> <br>
++    100 μL 100 mM ATP (Thermo #R0441) <br>
++     10 μL 1 M DTT <br>
++     ddH<sub>2</sub>O to 1 mL <br>
+Store at -20 °C in 40-60 μl aliquots. </div>
+<br>
+<div style="text-align: justify; margin-left:20px">
+. Add the following ingredients into a 0.2 mL tube in this orde rand vortex: </div>
+<div style="text-align: justify; margin-left:40px">
+linearized vector backbone (50 - 200 ng) <br>
++   each additional assembly piece (1:1 - 1:10 molar ratio of vector:insert) <br>
++   1 μL 10X SLiCE buffer <br>
++   1 μL PPY SLiCE extract <br>
++   ddH<sub>2</sub>O to 10 μL </div><br>
+<div style="text-align: justify; margin-left:20px">
+-> We use ratio vector:insert of 1:10 <br>
+-> Good to know: PPY in the strain whose cell extract is used for SLiCE reaction </div>
+<div style="text-align: justify; margin-left:20px">
+. Incubate the SLiCE reaction mix as above at 37 °C for 1 hour using a PCR machine or water bath, and then place on ice. <br>
+. Transform 1 - 10 μL of the assembly reaction into 50 μL of competent<i> E. coli </i> and/or run a diagnostic agarose gel to check for successful assembly. <br>
+Transformation of E. coli safer, but takes more time. <br>
+. For electroporation, transform 1 μL into 50 μL electrocompetent cells. For large recombinant DNA, electroporation is required. In complex cloning, electroporation is recommended, as it is 10-100 times as efficient as chemical transformation. <br>
+Electrocompetent cells have to be made, or we use heat shock (see protocol“transformationof <i>E. coli</i>”), protocol for electrocompetent cells can be taken from NEB</div>
+<br> <br>
+<hr size="10" noshade></hr>
-Zymo Research:
+<p style="font-size:12pt;"><sup>[1]</sup>
-Preparation of Competent Cells
+https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4672941/ (accessed 01 November 2017) </p>
-Grow  yeast cells  at 30
-°
-C in 10 ml YPD broth until mid-log phase (~5 x 10
- - 2 x 10
- cells/ml  or OD
- of  0.8-1.0).   The
-following steps are accomplished at room temperatur
-e.
-.    Pellet the cells at 500 x g for 4 minutes and di
-scard the supernatant.
-.   Add 10 ml
-EZ 1 solution
- to wash the pellet. Repellet the cells and discard
- the supernatant.
-.    Add 1 ml
-EZ 2 solution
-to resuspend the pellet.
-At this point, the competent cells can be used for
-transformations directly or stored frozen at or bel
-ow -70
-°
-C for future
-use.  It is important to freeze the cells slowly.
-To accomplish this, either wrap the aliquotted cell
-s in 2-6 layers of paper
-towels or place in a Styrofoam box before placing i
-n the freezer.
-DO NOT
- use liquid nitrogen to snap-freeze the cells.
-Transformation
-This  part  of  the  procedure  is  the  same  for  both  fro
-zen  stored  (thawed  at  room  temperature)  and  freshly
-  prepared
-competent yeast cells.
-.    Mix  50
-μ
-l  of  competent  cells  with  0.2-1
-μ
-g  DNA  (in less  than  5
-μ
-l  volume);  add  500
-μ
-l
-EZ  3  solution
-and mix thoroughly.
-.    Incubate  at  30
-°
-C  for  45  minutes.  Mix  vigorously  by  flicking  with  f
-inger  or  vortexing  (if  appropriate  for
-your DNA) 2-3 times during this incubation.
-.    Spread 50-150
-μ
-l of the above transformation mixture on an appropr
-iate plate.  It is unnecessary to pellet and
-wash the cells before spreading.
-Incubate the plates at 30
-°
-C for 2-4 days to allow for growth of transformants
-.
 </div></div>
@@ Line 841: / Line 1,160: @@
 <div class="spoiler">
-<input type="button"style="height: 50px; width: 50%; BACKGROUND-COLOR: #3399FF; font-size:25; color:black;" onclick="showSpoiler(this);" value="Ligation" />
+<input type="button" style="height: 50px; width: 50%; BACKGROUND-COLOR: rgb(61,67,80); font-size:25; color:white;" onclick="showSpoiler(this);" value="Transformation (E.Coli)" />
 <div class="inner" style="display:none;">
+<br> <br>
-Ligation Protocol with T4 DNA Ligase (M0202)
+<b>1. What is it?</b><br><br>
-.
+<div style="text-align: justify;">
-Set up the following reaction in a microcentrifuge tube on ice.
+Transmission of genetic information into competent cells (or plants, algas, mushrooms) (the target organismn)
-T4 DNA Ligase Bu
+</div><br>
-ff
+<b>2. Steps</b><br><br>
-er (10 x)
+<div style="text-align: justify; margin-left:20px">
-ul
+. Prechil 1.5 ml tube on ice. <br>
-T4 DNA Ligase
+. Thaw a tube competent <i>E. coli </i> cells on ice for 10 minutes. </div>
-ul
+<div style="text-align: justify; margin-left:40px">
-Vector DNA
+.  Mix gently. <br>
-Insert DNA
+. Pipette 50 µl of the cells  into the 1.5 ml tube. </div>
-Nuclease-free water
+<div style="text-align: justify; margin-left:20px">
-to 20 ul
+. Add 1-5 µl (containing 1 pg-100 ng of plasmid) DNA to the cell mixture. <br>
-.
+(as soon as as the last bit of ice in the tube is disappeared!)<br>
-Calculation of the DNA
+. Flick the tube 4-5 times to mix cells and DNA.
-.
+(No vortexing!)<br>
-Example calculation
+. Place the mixture on ice for 30 minutes.
-:3 vector to insert
+(without mixing!) <br>
-mass Vector DNA: 100 ng
+(2-fold loss in transformation efficiency for every 10 minutes this step is shortened!)
-Vector DNA: 10 kb
+<br>
-Insert DNA: 3 kb
+. Heat shock at exactly 42°C for exactly 30 seconds.
-.
+(without mixing!)<br>
-T4 DNA Ligase should be added last.
+(temperature and timing specific to transformation volume and vessel)<br>
-.
+. Place on ice for 5 minutes. <br>
-Use
-nebiocalculator.neb.com/#!/
- to calculate molar ratios.
-.
-The T4 DNA Ligase Bu
-ff
-er should be thawed and resuspended at room temperature.
-.
-Gently mix the reaction by pipetting up and down and microfuge briefly.
-.
-Incubation
-.
-cohesive (sticky) ends
-.
-°C overnight or room temperature for 10 minutes.
-.
-blunt ends or single base overhangs
-.
-°C overnight or room temperature for 2 hours
-(alternatively, high concentration T4
-DNA Ligase can be used in a 10 minute ligation)
-.
-.
-Heat inactivate at 65°C for 10 minutes.
-.
-Chill on ice and transform 1-5
-μ
-l of the reaction into 50
-μ
-l competent cells
-</div></div>
+(without mixing!)<br>
+. Pipette 950 µl of room temperature SOC into the mixture. <br>
+. Place at 37°C for 60 minutes and shake vigorously (800 rpm in thermo mix block).<br>
+(2-fold loss in transformation efficiency for every 15 minutes this step is shortened)<br>
+(SOC gives 2-fold higher transformation efficiency than LB medium)<br>
+(incubation with shaking or rotating the tube gives 2-fold higher transformation efficiency than without)<br>
+. Warm selection plates to 37 °C.<br>
+. Mix the cells thoroughly by flicking the tube and inverting. <br>
+. Spread 200 µl onto a selection plate and incubate overnight at 37 °C. <br>
+.For low efficiency cloning reactions: spin down the whole transformation mixture and remove the nearly complete supernatant (approx. 900 µl). Resuspend cells in remaining liquid and plate completely. </div><br><br>
+<hr size="10" noshade></hr>
+<p style="font-size:12pt;"><sup>[1]</sup>https://www.neb.com/protocols/1/01/01/high-efficiency-transformation-protocol-c2987 (accessed 01 November 2017)</p>
+ </div></div>
@@ Line 906: / Line 1,218: @@
 <div class="spoiler">
-<input type="button"  style="height:50px; width:50%; BACKGROUND-COLOR: 	#3399FF; font-size:25; color:black;" onclick="showSpoiler(this);" value="Miniprep"/>
+<input type="button" style="height: 50px; width: 50%; BACKGROUND-COLOR: rgb(61,67,80); font-size:25; color:white;" onclick="showSpoiler(this);" value="Transformation (yeast)" />
 <div class="inner" style="display:none;">
-<b>Promega “Wizard Plus SV Miniprep Purification System“</b>
 <br> <br>
-.Production of cleared lysate
+<b>1.Preparation of Competent Cells </b>
-<br>
+<br> <br>
-<p style="margin-left:30px;"> 1. Isolation of the bacteria </p>
+<div style="text-align: justify; margin-left:40px">1.Grow  yeast cells  at 30 °C in 10 ml YPD broth until mid-log phase <br>(~5 x 10<sup>6</sup> - 2 x 10<sup>7</sup> cells/ml  or OD <sub>600</sub> of  0.8-1.0).<br> The following steps are accomplished at room temperature. </div>
-<p style="margin-left:40px;">1.  harvest 1–5 ml (high-copy-number plasmid) or 10 ml (low-copy-number plasmid)
+<div style="text-align: justify; margin-left:40px">
-of bacterial culture
+.    Pellet the cells at 500 x g for 4 minutes and discard the supernatant. <br>
-<br>
+.   Add 10 ml EZ 1 solution to wash the pellet. Repellet the cells and discard the supernatant. <br>
-. centrifugation for 5 minutes at 10,000 xg in a tabletop centrifuge
+.    Add 1 ml EZ 2 solution to resuspend the pellet. At this point, the competent cells can be used for transformations directly or stored frozen at or below -70°C for future use.  It is important to freeze the cells slowly.  To accomplish this, either wrap the aliquotted cells in 2-6 layers of paper towels or place in a Styrofoam box before placing in the freezer.DO NOT use liquid nitrogen to snap-freeze the cells. </div><br>
-<br>
+<b>2. Transformation </b>
-. pour off the supernatant
+<br> <br>
-<br>
+<div style="text-align: justify; margin-left:20px">
-. reinsert again bacterial culture to the pellet and repeat step 2 and 3
+This  part  of  the  procedure  is  the  same  for  both  frozen  stored  (thawed  at  room  temperature)  and  freshly prepared competent yeast cells. </div>
-<br>
+<div style="text-align: justify; margin-left:40px">
-. blot the inverted tube on a paper towel to remove excess media
+.  Mix  50 μl  of  competent  cells  with  0.2-1 μg  DNA  (in less  than  5 μl  volume);  add  500 μl EZ 3 solution and mix thoroughly. <br>
-<br> </p>
+. Incubate  at  30 °C  for  45  minutes.  Mix  vigorously  by  flicking  with  finger  or  vortexing  (if appropriate  for your DNA) 2-3 times during this incubation. <br>
-.
+.    Spread 50-150
-Resuspension
+μl of the above transformation mixture on an appropriate plate.  It is unnecessary to pellet and wash the cells before spreading. Incubate the plates at 30°C for 2-4 days to allow for growth of transformants. </div>
-of the cells
+<br> <br>
-.
+<hr size="10" noshade></hr>
-add 250
-μ
+<p style="font-size:12pt;"><sup>[1]</sup>
-l of Cell Resuspension Solution
+http://www.zymoresearch.com/downloads/dl/file/id/165/t2001i.pdf (accessed 01 November 2017)</p>
-.
+</div></div>
-completely resuspend the cell pellet by vortexing or pipetting
-.
-it is essential to thoroughly resuspend the cells
-.
-Lysing
-.
-add 250
-μ
-l of Cell Lysis Solution
-.
-mix by inverting the tube 4 times -
-do not vortex
-.
-incubate until the cell suspension clears (clear
-≠
-  colorlessly) (
-approximately 1–
-minutes
-)
-.
-  Splitting proteins
-.
-add 10
-μ
-l of Alkaline Protease Solution
-.
-mix by inverting the tube 4 times -
-do not vortex
-.
-incubate for 5 minutes
-at room temperature
-.
-Neutralization
-.
-add 350
-μ
-l of Neutralization Solution
-.
-immediately mix by inverting the tube 4 times -
-do not vortex
-.
-Isolation of the plasmids
-.
-centrifuge the bacterial lysate at maximum speed (around 14,000
-×
-g
-) in a
-microcentrifuge for 10 minutes at room temperature
- </div></div>
@@ Line 990: / Line 1,256: @@
          </div>
      </div>
+</div>
@@ Line 1,042: / Line 1,308: @@
          </div>
      </div>
 </div>