Line 592: | Line 592: | ||
*1000x stock chloramphenicol (25mg/ml) | *1000x stock chloramphenicol (25mg/ml) | ||
== Procedure: == | == Procedure: == | ||
− | + | #Pellet 1–5 ml bacterial culture by centrifugation for 30 seconds. Discard supernatant. | |
− | + | #Re-suspend pellet in 200 μl Plasmid Resuspension Buffer (B1). Vortex or pipet to ensure cells are completely resuspended. There should be no visible clumps. | |
− | + | #Add 200 μl Plasmid Lysis Buffer (B2), gently invert tube 5–6 times, and incubate at room temperature for 1 minute. Colour should change to dark pink, and solution will become transparent and viscous. Do not vortex. | |
− | + | #Add 400 μl of Plasmid Neutralisation Buffer (B3), gently invert tube until neutralised, and incubate at room temperature for 2 minutes. Sample is neutralised when colour is uniformly yellow and precipitate forms. Do not vortex. | |
− | + | #Centrifuge lysate for 2–5 minutes. For culture volumes >1 ml, we recommend a 5 minute spin to ensure efficient RNA removal by RNase A. Pellet should be compact; spin longer if needed. | |
− | + | #Carefully transfer supernatant to the spin column and centrifuge for 1 minute. Discard flow-through. | |
− | + | #Re-insert column in the collection tube and add 200 μl of Plasmid Wash Buffer 1. Centrifuge for 1 minute. Discarding the flow-through is optional. | |
− | + | #Add 400 μl of Plasmid Wash Buffer 2 and centrifuge for 1 minute. | |
− | + | #Transfer column to a clean 1.5 ml microfuge tube. Use care to ensure that the tip of the column does not come into contact with the flow-through. If there is any doubt, re-spin the column for 1 minute. | |
− | + | #Add ≥ 30 μl DNA Elution Buffer to the centre of the matrix. Wait for 1 minute, then spin for 1 minute at 16,000 x g to elute the DNA. Nuclease-free water (pH 7–8.5) can also be used to elute the DNA. | |
SOURCE: [https://international.neb.com/protocols/2015/11/20/monarch-plasmid-dna-miniprep-kit-protocol-t1010 New England Biolabs] | SOURCE: [https://international.neb.com/protocols/2015/11/20/monarch-plasmid-dna-miniprep-kit-protocol-t1010 New England Biolabs] | ||
Line 619: | Line 619: | ||
''At least one enzyme has < 100% activity in this buffer, so additional units of enzyme and/or longer incubation time may be necessary.'' | ''At least one enzyme has < 100% activity in this buffer, so additional units of enzyme and/or longer incubation time may be necessary.'' | ||
− | + | {{{!}} | |
− | + | ! Enzyme: {{!}}{{!}} Temp: (*C) {{!}}{{!}} %Activity in NEBuffer | |
− | + | {{!}}- | |
− | + | {{!}} {{!}}{{!}} {{!}}{{!}} 1.1 {{!}}{{!}} 2.1 {{!}}{{!}} 3.1 | |
− | + | {{!}}- | |
− | { | + | {{!}} EcoRI-HF {{!}}{{!}} 37 {{!}}{{!}} 10 {{!}}{{!}} 100 {{!}}{{!}} 10 |
− | ! Enzyme: | + | {{!}}- |
− | + | {{!}} Pstl {{!}}{{!}} 37 {{!}}{{!}} 75 {{!}}{{!}} 75 {{!}}{{!}} 100 | |
− | + | {{!}}} | |
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
== Materials: == | == Materials: == | ||
Line 649: | Line 644: | ||
# Set up the following reaction (total reaction volume 20 μl). | # Set up the following reaction (total reaction volume 20 μl). | ||
− | { | + | {{{!}} |
− | + | {{!}} A {{!}}{{!}} B | |
− | + | {{!}}- | |
− | + | {{!}}Material {{!}}{{!}}Reagent volumes (ul) | |
− | + | {{!}}- | |
− | + | {{!}} Buffer (10x) {{!}}{{!}} 2 | |
− | + | {{!}}- | |
− | + | {{!}} DNA * {{!}}{{!}} 10 | |
− | + | {{!}}- | |
− | + | {{!}} Restriction enzyme EcoR1 {{!}}{{!}} 1 | |
− | + | {{!}}- | |
− | + | {{!}} Restriction enzyme Pstl {{!}}{{!}} 1 | |
− | + | {{!}}- | |
− | + | {{!}} Nuclease free water {{!}}{{!}} 6 | |
− | + | {{!}}- | |
− | + | {{!}} Total Volume (ul) {{!}}{{!}} 20 | |
− | + | {{!}}} | |
− | |||
* Recommended maximum of 1 μg of substrate per 10 units of enzyme. ** Restriction Enzymes should be added to the mixture last. | * Recommended maximum of 1 μg of substrate per 10 units of enzyme. ** Restriction Enzymes should be added to the mixture last. | ||
Line 674: | Line 668: | ||
# Incubate for 1 hour at the enzyme-specific appropriate temperature. Then heat inactivate at 80°C. | # Incubate for 1 hour at the enzyme-specific appropriate temperature. Then heat inactivate at 80°C. | ||
# PCR purify at the end to remove the short fragments. | # PCR purify at the end to remove the short fragments. | ||
− | |||
}} | }} | ||
Line 680: | Line 673: | ||
{{CLSB-UK Protocol | {{CLSB-UK Protocol | ||
|title=Characterising araC-Pbad Promoter | |title=Characterising araC-Pbad Promoter | ||
− | |||
|content= | |content= | ||
Line 689: | Line 681: | ||
{{CLSB-UK Protocol | {{CLSB-UK Protocol | ||
|title=Characterising 15GFP Toehold Switch | |title=Characterising 15GFP Toehold Switch | ||
− | |||
|content= | |content= | ||
Revision as of 12:02, 29 October 2017
Contents
- 1 Experiments
- 1.1 Methodology
- 1.2 Protocols
- 1.3 Introduction
- 1.4 Materials
- 1.5 Procedure
- 1.6 Materials
- 1.7 Procedure
- 1.8 Introduction
- 1.9 Materials
- 1.10 Procedure
- 1.11 Single-temperature Double digests for gBlocks
- 1.12 Introduction
- 1.13 Materials
- 1.14 Procedure
- 1.15 Single-temperature double digest of BBa_J04450
- 1.16 Introduction
- 1.17 Materials
- 1.18 Procedure
- 1.19 Introduction
- 1.20 Materials
- 1.21 Procedure
- 2 Ligation with T4 DNA Ligase and ara-gBlocks
Experiments
Methodology
Protocols
Amplifying gBlocks using PCR
Introduction
gBlock Gene Fragments are normalised to 250 or 1000 ng, depending on length, which is a suitable quantity for many applications. However, we want to amplify gBlocks Gene Fragments to get additional starting material.
- GFP/ Luciferase 15b/27b - 1000ng
- 15b/27b - 250ng
Materials
gBlock Gene Fragments Amplification primers Phusion DNA Polymerase (www.NEB.com/ Phusion) PCR or gel purification kit (depending on application)
Procedure
Resuspening gBlocks
- The dried down gBlocks Gene Fragment pellet can become displaced from the bottom of the tube during shipping. Centrifuge the tube for 3−5 sec at a minimum of 3000 x g to pellet the material to the bottom of the tube.
- Add TE to the tube for your desired final concentration
- Briefly vortex and centrifuge
- Store at -20°C for up to 24 months
Resuspension volume of TE buffer (μL) | ||
---|---|---|
gBlock gene fragment final concentration | 15b/27b toehold switch (250ng) | GFP switch (1000ng) |
10 ngμL | 25 | n/a |
20 ngμL | n/a | 50 |
Resuspending the primers for PCR
- Centrifuge the tube for 3−5 seconds at a minimum of 3000 x g to pellet the material to the bottom of the tube.
- To make a 100 μM concentration: Take the number of nmoles of oligo in the tube and multiply that by 10. This number will be the volume (μL) of buffer to add to get a 100 μM solution.
- Store resuspended oligonucleotides as several small aliquots at –20°C.
Amplifying gBlocks
- A precipitate (most noticeable after the first 1–2 freeze/thaw cycles) is not uncommon. To ensure optimal performance, the master mix should be thawed and resuspended prior to use. Stability testing using up to 15 freeze/thaw cycles has shown no negative effect on master mix performance.
- Dilute the gBlocks 15b/27b by using 1uL of the stock and 9uL of the TEB buffer. For other blocks use 1μL of the block and 19uL of the TEB buffer. This will give us both in concentrations of 1 ng/μL. Dilute the primers 1:10 to get 10μM concentration.
- Assemble all reaction components on ice and quickly transfer the reactions to a thermocycler preheated to the denaturation temperature (98°C). All components should be mixed prior to use.
Component | Volume for the 25μL reaction (μL) | Volume for the 25μL reaction (μL) |
GFP | Basic blocks | |
Q5 High-Fidelity 2X Master Mix | 12.5 | 12.5 |
10uM forward primer | 1.25 | 1.25 |
10uM reverse primer | 1.25 | 1.25 |
Template DNA (1ng) | 1 | 1 |
Nuclease-free water | 9 | 9 |
- Gently mix the reaction. Collect all liquid to the bottom of the tube by a quick spin if necessary.
- Transfer PCR tubes to a thermocycler and begin thermocycling (Programme 226).
Step | Temperature (℃) | Time (s) |
Initial denaturing | 98 | 30 |
25 cycles | 98 | 10 |
62 | 20 | |
72 | 30 | |
Final extension | 72 | 300 |
Hold | 4 | Indefinite |
The temperatures and times which the IDT document suggested for the thermocycling procedure were not successful at first. Our yield was lower than expected and this was due to “unclean” amplification where there was lots of non-specific primer binding. In an attempt to overcome this, we tried many different temperatures and timings. The table above illustrates the combination which produced the best results and gave us the highest yield.
The primers we used for our PCR reactions were designed using a primer tool on the Benchling website.
Source: IDT
Gel Electrophoresis
Materials
- Gel Box
- Gel Tray
- Comb
- 1% Agarose in 1X TAE
- SYBR Safe
- 100ml beaker
- Pipettes and pipette tips
Procedure
Preparing the Gel
- Prepare 300ml of the TAE buffer by dissolving 6ml of the 50X concentrated buffer in 294ml of distilled water.
- Add 0.5g of agarose powder into 5ml of the diluted buffer and heat whilst stirring continually until clear.
- Pour into the gel mould and add the comb with a required number of wells.
- Pipette in 3uL of SYBR Safe dye and use the pipette tip to mix it around.
Preparing the material for loading
- Mix 3uL of the DNA sample with 2uL of the loading dye.
Running the gel
- Add enough 1X TAE to fill the reservoirs at both ends of the gel box and cover the surface of the gel - the gel should be immersed. You should use the remaining TAE buffer from 300ml.
- Load 1μL prepared ladder (+dye +loading buffer). The concentration is 100μg/ml, i.e. there will be a total of 0.1ug = 100ng of ladder in the well. The mass of ladder is important to know if you need to quantify your bands by comparison with the ladder bands.
Load ladder in left-most lane.
- Use 2 μL loading dye per 3 μL of sample.
- Load samples left to right.
The capacity of the 8 well, 1.5mm wide well is approximately 45 μL. The capacity of the 15 well, 1.5mm wide well is approximately 15 μL.
- Place cover on gel box such that your samples will run towards the positive, red electrode. Make sure that the cables from the cover are connected to the power supply correctly.
- Turn on the power supply and run your gel at ~85 V for 1 hr 20 mins (voltage and time values can vary). Check regularly to see how far the dye has moved and stop the gel when the dye has moved approximately 80% of the distance.
- Verify that bubbles are rising from the electrodes once you start your gel to ensure your gel is running properly.
SOURCE: [http://www.openwetware.org/wiki/Agarose_gel_electrophoresis OpenWetWare]
PCR Cleanup
Introduction
Input amount of DNA to be purified should not exceed the binding capacity of the column (5 μg). A starting sample volume of 20–100 μl is recommended. For smaller samples, TE can be used to adjust the volume to the recommended volume range. Centrifugation should be carried out at 16,000 x g in a standard laboratory microcentrifuge at room temperature.
Materials
- Monarch® PCR & DNA Cleanup Kit
- DNA samples from PCR reaction
Procedure
Cleanup Procedure
- Add ethanol to Monarch DNA Wash Buffer prior to use (4 volumes of ≥ 95% ethanol per volume of Monarch DNA Wash Buffer).
- Dilute sample with DNA Cleanup Binding Buffer according to the table below. Mix well by pipetting up and down or flicking the tube. Do not vortex. A starting sample should include combined products of PCR amplification, i.e. 72uL for GFP15b/GFP27b/15b/27b
| Ratio of binding buffer : sample | - | | 5:1 | }
Double digests of Vectors and InsertsSingle-temperature Double digests for gBlocksIntroductionThis protocol was set up whilst waiting to see if we managed to successfully isolate the white colonies from the previous ligations. As PstI could have been the problem, we decided to try cloning the gBlocks into the plasmid using the EcoRI and SpeI instead and this combination of enzymes proved more successful that EcoRI and PstI.
Double Digest Recommendations for EcoRI-HF® + SpeI: Digest in NEBuffer 2.1 at 37°C. At least one enzyme has < 100% activity in this buffer, so additional units of enzyme and/or longer incubation time may be necessary.
Materials
ProcedureSingle temperature double digest reaction
Alternatively to the first 2 steps, use the gBlocks from the PCR amplification reaction.
Single-temperature double digest of BBa_J04450IntroductionWe previously tried using a linearised plasmid backbone but were not successful at identifying correctly ligated constructs. This prompted us to use BBa_J04450. We need to digest enough of the BBa_J04450 plasmid for 4 reactions as we have 4 parts. This is the Double Digest Protocol with EcoRI-HF and PstI, using a common reaction and same incubation temperature for both enzymes. Double Digest Recommendations for EcoRI-HF® + PstI: Digest in NEBuffer 2.1 at 37°C. At least one enzyme has < 100% activity in this buffer, so additional units of enzyme and/or longer incubation time may be necessary.
Materials
Before the procedure aliquot all the enzymes and buffer first to prevent freeze-thaw in the future.
ProcedureSingle-temperature double digest reaction
Ligation with T4 DNA LigaseIntroductionThis was the protocol we used for the first set of gBlocks with constitutive promoters. Materials
ProcedureSet-up the T4 DNA Ligase ReactionNote: T4 DNA Ligase should be added last. The table shows a ligation using a molar ratio of 1:3 vector to insert for the indicated DNA sizes.
Tip: Aliquot the 10x buffer less concentrated so when thawing, the DTT gets soluble more easily.
Ligation with T4 DNA Ligase and ara-gBlocksIntroductionThis was the protocol we used for the new set of gBlocks which had arabinose promoters. We decided to discontinue the use of Luciferase gBlocks at this point in time. Materials
ProcedureSet-up the T4 DNA Ligase ReactionVector concentration - 231ng from the digestion reaction which underwent PCR cleanup into 16uL of nuclease free water, making the concentration 14.4ng/uL. To get 30ng we need 2.1 uL of this stock. 15b/27b concentration - from the gel 15ng/ul. Use 1uL per reaction. GFP15/GFP27 concentration - we digested 300ng of gBlocks and this underwent PCR cleanup into 16uL giving approximate concentration of 18ng/uL. To get 90 ng, we need 6uL of the block. Note: T4 DNA Ligase should be added last. The table shows a ligation using a molar ratio of 1:3 vector to insert for the indicated DNA sizes.
Tip: Aliquot the 10x buffer less concentrated so when thawing, the DTT gets soluble more easily.
|