PCR Protocols
PCR
What you need:
- A container of ice
- Small PCR tubes
- Template DNA containing the gene of interest
- DNA primers designed to amplify the gene of interest
- Phusion DNA polymerase
- dNTP solution
- DMSO (optional)
- 5X HF or GC buffer
- Autoclaved MilliQ water
Procedure:
1. Keep all components on ice as you work
2. Label clearly one small PCR tube for each sample you wish to run.
3. For each sample, mix the following:
- 0.75 uL template DNA
- 1uL each of the two primers
- 0.5uL DNA polymerase
- 1uL dNTPs
- 1.5 uL DMSO (optional)
- 10uL 5X HF/CG buffer
- 33.5 uL autoclaved water
4. Use your pipet to mix thoroughly
5. Load samples in the PCR machine and select the phusion protocol, using an extension time of roughly one minute per kb of DNA to be amplified. However – consult the bootcamp optimized PCR protocol if necessary.
Note: When running PCR with the supermix use 25 uL of supermix, 1.5 uL of DMSO (optional), 0.5uL DNA polymerase, 1uL each of the two primers, 0.75 uL of template, and autoclaved water to 50uL volume.
PCR Cleanup
What you need:
- Large Zymo-spin columns in collection tubes
- The completed PCR of interest
- DNA binding buffer
- DNA wash buffer
- DNA wash buffer
- Autoclaved 1.5 mL centrifuge tubes
- Autoclaved MilliQ water
Procedure:
1. To the PCR liquid, add 2 volumes of DNA binding buffer
2. Mix briefly by pipetting and add all of the mixture to a large Zymo-spin column
3. Centrifuge at full speed for 30 seconds
4. Discard the waste liquid in the collection tube
5. To the column, add 200 uL wash buffer
6. Centrifuge for 30 seconds and discard the waste liquid
7. Repeat steps 5-6
8. Centrifuge the empty column for 1 minute
9. Replace the collection tube with 1.5 mL centrifuge tube
10. Gently add 30uL of autoclaved water to the column
11. Let the column incubate at room temperature for 2 minutes
12. Centrifuge 1 minute
13. Discard the column
14. Label the collected liquid and store it in the -20˚C freezer until use
PCR Troubleshooting
Ran a diagnostic test to optimize buffer and temperature for the PCR. After doing the PCR (it’s nice to do this in linked PCR tubes) we run on a gel to test which worked best.
Buffers tested: GC + DMSO, HF + DMSO, GC, buffer G supermix + DMSO (3 tubes of each)
Made a master mix of primers, phusion, template DNA, and dNTPs. This was then used this for all the tubes of GC+DMSO, HF+DMSO, and GC.
Master Mix:
- 10 uL dNTP’s
- 2 uL forward primers
- 2 uL reverse primers
- 2 uL template DNA
- 1 uL phusion
GC + DMSO:
- 18 uL H2O
- 6 uL GC buffer
- 5.1 uL of master mix
- 0.9 uL DMSO
- now divide into 3 tubes (10 uL into each PCR tube)
HF + DMSO:
- 18 uL H2O
- 6 uL HF buffer
- 5.1 uL of master mix
- 0.9 uL DMSO
Now divide into 3 tubes (10 uL into each PCR tube)
GC:
- 18.9 uL H2O
- 6 uL GC buffer
- 5.1 uL master mix
Now divide into 3 tubes (10 uL into each PCR tube)
Supermix:
- 15 uL buffer G
- 12 uL H2O
- 0.9 uL DMSO
- 0.6 uL forward primer
- 0.6 uL reverse primer
- 0.6 uL template DNA
- 0.3 uL phusion
PCR program:
1. 98 degrees Celsius for 3 minutes
2. 98 degrees C for 15 seconds
3. Temperature gradient 48-55-63 degrees for 30 seconds
4. 72 degrees C for 1 minute
5. Go to step 2, repeat 30 times
6. 72 degrees C for 5 minutes
Gel results: Only buffer G +DMSO works and it works at all temperatures.
Optomized PCR
Worked using buffer G +DMSO supermix at 63o C.
Supermix (for three reactions. 10uL/rxn):
- 15 uL buffer G
- 12 uL H2O
- 0.9 uL DMSO
- 0.6 uL forward primer
- 0.6 uL reverse primer
- 0.6 uL template DNA
- 0.3 uL phusion
PCR program (running time of just under 2 hours):
1. 98 degrees Celsius for 3 minutes
2. 98 degrees C for 15 seconds
3. 63 degrees C for 30 seconds
4. 72 degrees C for 1 minute
5. Go to step 2, repeat 30 times
6. 72 degrees C for 5 minutes
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