Phusion colony PCR
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Colony PCR of yeast cells using Phusion
This is a slightly slower and more expensive protocol than the TopTaq protocol, but also more robust for longer or more complex amplicons. For E. coli see here.
Prepare mastermix:
Do sequencing reaction: | |
---|---|
Phusion HF Buffer 5X | 5 μl |
H2O | 12 μl |
dNTPs (10 mM each) | 1 μl |
Primer Fwd (10 μM) | 1 μl |
Primer Rev (10 μM) | 1 μl |
Divide over PCR reaction tubes/wells and add some fresh yeast cells to the mix using pipette tips (so not toothpicks).
Boil the sample in the PCR machine at 98°C for 10 minutes and cool down to 7°C. Now add to each tube 5μl of a mix of:
- 0.2 μl Phusion polymerase
- 5 μl H2O
We dilute to Phusion to make the pipetting a bit easier and obtain the right final concentration for all ingredients.
Run using program (bart -> pombe -> colony PCR)
Number of cycles | Temperature | Duration |
---|---|---|
1 X | 94°C | 2 min |
35X | 94°C | 30 sec |
55-60°C | 30 sec | |
72°C | 2 min 30 sec | |
1X | 72°C | 7 min |
7°C | Inf |