Paulinchen (Talk | contribs) (buttons farben) |
Paulinchen (Talk | contribs) |
||
Line 51: | Line 51: | ||
<div class="spoiler"> | <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: #3399FF; font-size:25; color:black;" onclick="showSpoiler(this);" value="Colony PCR" /> | ||
− | <div class="inner" style="display:none;"> | + | <div class="inner" style="display:none;"> |
+ | |||
+ | <b>Colony PCR with ALLin™ Red Taq Mastermix, 2X: </b> | ||
+ | <br> <br> | ||
+ | <b>aim: </b><br> | ||
+ | Is the insert DNA in the plasmid present or absent? <br> | ||
+ | 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:10px;">- resuspend colonies: <br> | ||
+ | - label PCR tubes or wells of a PCR plate <br> | ||
+ | - pipette 10 – 20 μl PCR grade water into each tube/well <br> | ||
+ | - transfer transformants using sterile pipette or toothpicks into the individual tubes/wells <br> | ||
+ | - the amount of cells resuspended must just be visible <br> | ||
+ | - Resuspend each colony by stirring with the tip or toothpick. </div> <br> | ||
+ | - Prepare a PCR master mix (always prepare at least 10 % more, use the excel sheet by Sophia | ||
+ | to calculate) <br> | ||
+ | - 20 μl would be good, Fabian suggested 5 μl <br> | ||
+ | Mix gently, avoid bubbles. <br> | ||
+ | - Aliquote the 22.5 μl of PCR master mix into each PCR tube <br> | ||
+ | - Add 2.5 μl of the resuspended colony or overnight culture <br> | ||
+ | <br> | ||
+ | Do not forget the negative control! <br> | ||
+ | <br> | ||
+ | - Close tube <br> | ||
+ | - Perform the PCR using Thermocycler as follow: <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-031e">Initial denaturation<br></th> | ||
+ | <th class="tg-yw4l">1 cycle<br></th> | ||
+ | <th class="tg-yw4l">95°C</th> | ||
+ | <th class="tg-yw4l">60s</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td class="tg-yw4l">Denaturation</td> | ||
+ | <td class="tg-yw4l">30-40 cycles<br></td> | ||
+ | <td class="tg-yw4l">95°C</td> | ||
+ | <td class="tg-yw4l">15s</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td class="tg-yw4l">Annealing</td> | ||
+ | <td class="tg-yw4l">30-40 cycles<br></td> | ||
+ | <td class="tg-yw4l">55-65°C</td> | ||
+ | <td class="tg-yw4l">15s</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td class="tg-yw4l">Extension</td> | ||
+ | <td class="tg-yw4l">30-40 cycles<br></td> | ||
+ | <td class="tg-yw4l">72°C</td> | ||
+ | <td class="tg-yw4l">15-90s</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td class="tg-yw4l">Final extension<br></td> | ||
+ | <td class="tg-yw4l">1 cycle<br></td> | ||
+ | <td class="tg-yw4l">72°C</td> | ||
+ | <td class="tg-yw4l">5 min<br></td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <br> <br> | ||
+ | - Store probes for short time on ice, for long at -20°C <br> | ||
+ | - Load probes on the agarose gel e.g. 10 μl (so in case you have enough left for another round). <br> | ||
+ | <br> | ||
+ | |||
+ | <b>step by step for yeast: </b> | ||
+ | - 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. | ||
+ | If overnight cultures are to be used: pipette 40 μl of a 0.1 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. | ||
+ | 2. | ||
+ | Prepare a PCR master mix (always prepare at least 10% more, use the excel sheet to | ||
+ | calculate) | ||
+ | 3. | ||
+ | Aliquot 22.5 μl of PCR master mix into each PCR tube. | ||
+ | 4. | ||
+ | Add 2.5 μl of the resuspended colony or overnight culture mixed with NaOH to the | ||
+ | appropriate PCR tube. | ||
+ | 5. | ||
+ | Close the tubes. | ||
+ | 6. | ||
+ | Perform the PCR using the following cycling profle: | ||
+ | * | ||
+ | NaOH opens the cells. | ||
+ | 7. | ||
+ | Load probes on the agarose gel. | ||
+ | 8. | ||
+ | Store probes for short time on ice, for long at -20°C. | ||
+ | |||
+ | </div></div> | ||
+ | |||
+ | |||
Line 65: | Line 173: | ||
<div class="spoiler"> | <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: #3399FF; font-size:25; color:black;" onclick="showSpoiler(this);" value="Gel electrohoresis" /> | ||
− | <div class="inner" style="display:none;"> Protocol is | + | <div class="inner" style="display:none;"> |
+ | |||
+ | Gel Electrophoresis | ||
+ | What is it ? | ||
+ | – | ||
+ | standard lab procedure for separating DNA by size (e.g., length in base pairs) for | ||
+ | visualization and purification | ||
+ | – | ||
+ | uses an electrical field to move the negatively charged DNA through an agarose gel matrix | ||
+ | toward a positive electrode. | ||
+ | Shorter DNA fragments migrate through the gel more quickly than longer ones. | ||
+ | Why are we doing it ? | ||
+ | – | ||
+ | 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). | ||
+ | Protocol : Agarose Gel Electrophoresis | ||
+ | Pouring a Standard 1% Agarose Gel: | ||
+ | 1. | ||
+ | M | ||
+ | easure 1g agarose and and mix it with 100ml 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). | ||
+ | 1L 10x stock TBE: in the lab | ||
+ | Note: 10x stock of TBE has to be diluted to a 1x puffer! | ||
+ | 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. | ||
+ | Note: | ||
+ | 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. | ||
+ | Pouring of the gel | ||
+ | 4. 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 | ||
+ | 5. | ||
+ | (Optional) | ||
+ | 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: Get a Safety Briefing from Fabian or Lena before working with EtBr !!! | ||
+ | Note: | ||
+ | Caution EtBr is a known mutagen. Wear a lab coat, eye protection and gloves when working | ||
+ | with this chemical. | ||
+ | Note: | ||
+ | If you add EtBr to your gel, you will also want to add it to the running buffer when you run | ||
+ | the gel. | ||
+ | 6. | ||
+ | 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.) | ||
+ | Note: | ||
+ | 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. | ||
+ | 7. Let the newly poured gel sit at room temperature for 20-30 mins, until it has completely | ||
+ | solidified. | ||
+ | Note: | ||
+ | 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. | ||
+ | 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: | ||
+ | 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. | ||
+ | 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. | ||
+ | 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. | ||
+ | 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 | ||
+ | |||
+ | </div></div> | ||
+ | |||
+ | |||
Line 71: | Line 302: | ||
<div class="spoiler"> | <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: #3399FF; font-size:25; color:black;" onclick="showSpoiler(this);" value="PCR" /> | ||
− | <div class="inner" style="display:none;"> | + | <div class="inner" style="display:none;"> |
+ | |||
+ | What is the PCR ? | ||
+ | Method to make multiple copies of a | ||
+ | the specific DNA-sequence | ||
+ | Protocol for PCR with Q5 High- Fidelity 2x Master Mix | ||
+ | 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. | ||
+ | Reaction Setup: | ||
+ | – | ||
+ | assemble all reaction components on ice, work on ice while assembling | ||
+ | – | ||
+ | preheat the thermocycler to the denaturation temperature( 98 °C) | ||
+ | – | ||
+ | prior to use all components should be mixed | ||
+ | – | ||
+ | work quickly when transferring the reactions to a thermocycler | ||
+ | 1. | ||
+ | on ice | ||
+ | Assemble all components for the reaction : | ||
+ | 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 | ||
+ | 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. | ||
+ | 2. Transfer PCR tubes to a PCR machine and begin thermocycling. | ||
+ | Steps | ||
+ | of | ||
+ | PCR: | ||
+ | 1.Denaturation : double- stranded template DNA is heated to separate it into two single stands | ||
+ | 2. Annealing : temperature is lowered to enable the DNA primers to attach to the template DNA | ||
+ | 3. 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 | ||
+ | 98°C | ||
+ | 30 seconds | ||
+ | 25–35 Cycles | ||
+ | 98°C | ||
+ | 5–10 seconds | ||
+ | *50–72°C | ||
+ | 10–30 | ||
+ | seconds | ||
+ | 72°C | ||
+ | 20–30 | ||
+ | seconds | ||
+ | /kb | ||
+ | Final Extension | ||
+ | 72°C | ||
+ | 2 | ||
+ | minutes | ||
+ | Hold | ||
+ | 4–10°C | ||
+ | hold is not | ||
+ | necessary | ||
+ | 1. | ||
+ | Template: | ||
+ | Use of high quality, purified DNA templates greatly enhances the success of PCR. | ||
+ | Recommended amounts of DNA template for a 50 μl reaction are as follows: | ||
+ | DNA | ||
+ | AMOUNT | ||
+ | DNA Genomic | ||
+ | 1 ng–1 μg | ||
+ | Plasmid or Viral | ||
+ | 1 pg–1 ng | ||
+ | 2. | ||
+ | Primers: | ||
+ | Oligonucleotide primers are generally 20–40 nucleotides in length and ideally have a GC | ||
+ | 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. | ||
+ | 3. | ||
+ | Mg | ||
+ | ++ | ||
+ | and additives: | ||
+ | The | ||
+ | Q5 High-Fidelity Master Mix contains 2.0 | ||
+ | mM Mg | ||
+ | ++ | ||
+ | when used at a 1X concentration. | ||
+ | This is optimal for most PCR products generated with this master mix. | ||
+ | 4. | ||
+ | Deoxynucleotides: | ||
+ | The final concentration of dNTPs is 200 μM of each deoxynucleotide in the 1X | ||
+ | Q5 High- | ||
+ | Fidelity Master Mix. | ||
+ | Q5 High-Fidelity DNA Polymerase cannot incorporate dUTP and is not | ||
+ | recommended for use with uracil-containing primers or templates. | ||
+ | 5. | ||
+ | Q5 | ||
+ | High-Fidelity DNA Polymerase concentration: | ||
+ | The concentration of | ||
+ | Q5 High-Fidelity DNA Polymerase in the | ||
+ | Q5 High-Fidelity 2X Master | ||
+ | Mix has been optimized for best results under a wide range of conditions. | ||
+ | 6. | ||
+ | Denaturation: | ||
+ | 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. | ||
+ | 7. | ||
+ | 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). | ||
+ | 8. | ||
+ | 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. | ||
+ | 9. | ||
+ | Cycle number: | ||
+ | Generally, 25–35 cycles yield sufficient product. | ||
+ | For genomic amplicons, 30-35 cycles are | ||
+ | recommended. | ||
+ | 10. | ||
+ | 2-step PCR: | ||
+ | When primers with annealing temperatures ≥ | ||
+ | 72°C are used, a 2-step thermocycling protocol | ||
+ | (combining annealing and extension into one step) is possible. | ||
+ | 11. | ||
+ | Amplification of long products: | ||
+ | When amplifying products > 6 kb, it is often helpful to increase the extension time to 40–50 | ||
+ | seconds/kb. | ||
+ | 12. | ||
+ | PCR product: | ||
+ | The PCR products generated using | ||
+ | Q5 High-Fidelity | ||
+ | 2X | ||
+ | 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> | ||
+ | |||
+ | |||
Revision as of 19:19, 25 October 2017
Our research work
We are describing our research work. Below you can find the protocols we used.
Protocols
Main sponsors:
![](https://static.igem.org/mediawiki/2017/f/fd/T--Potsdam--sponors--up_unten.png)
![](https://static.igem.org/mediawiki/2017/8/8d/T--potsdam--sponsors--maxsynbio_unten.png)
![](https://static.igem.org/mediawiki/2017/8/8f/T--Potsdam--sponsors--promega_unten.png)
![](https://static.igem.org/mediawiki/2017/b/b2/T--Potsdam--sponsors--eppendorf.png)