Team:UIUC-Illinois/Notebook/Protocols/PCR

Select Protocol

• Bootcamp Protocols
• Subculturing Plates
• Making Electrocompetent E.Coli
• Making TAE Buffers
• Making Electrophoresis Gels
• Gel Purification
• Inoculation
• Miniprep
• PCR Protocols
• Digestions
• Ligations
• Transformation of E.Coli
• Storage of Cells

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