Difference between revisions of "Phusion colony PCR"
From PombEvolution
Jump to navigationJump to search| Line 2: | Line 2: | ||
== Colony PCR of yeast cells using Phusion == | == Colony PCR of yeast cells using Phusion == | ||
| − | This is a slightly slower and '''more expensive''' protocol than the [[Yeast Colony PCR | + | This is a slightly slower and '''more expensive''' protocol than the [[Yeast Colony PCR|TopTaq protocol]], but also more robust for longer or more complex amplicons. For ''E. coli'' see [[E_coli_colony_PCR| here]]. <BR> |
| − | + | Prepare mastermix: | |
| − | <BR>Prepare mastermix: | ||
{| class="wikitable" | {| class="wikitable" | ||
!Do sequencing reaction: | !Do sequencing reaction: | ||
! | ! | ||
|- | |- | ||
| − | | Phusion HF Buffer 5X || | + | | Phusion HF Buffer 5X || 5 μl |
|- | |- | ||
| − | |H<sub>2</sub>O|| | + | |H<sub>2</sub>O||12 μl |
|- | |- | ||
| − | |dNTPs (10 mM each)|| | + | |dNTPs (10 mM each)|| 1 μl |
|- | |- | ||
| − | |Primer Fwd (10 μM) || | + | |Primer Fwd (10 μM) || 1 μl |
|- | |- | ||
| − | |Primer Rev (10 μM) || | + | |Primer Rev (10 μM) || 1 μl |
|} | |} | ||
| − | Divide over PCR reaction tubes/wells | + | Divide over PCR reaction tubes/wells and add some fresh yeast cells to the mix using pipette tips (so '''not''' toothpicks). <BR> |
| + | |||
| + | |||
Boil the sample in the PCR machine at 98°C for 10 minutes and cool down to 7°C. | 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: | Now add to each tube 5μl of a mix of: | ||
* 0.2 μl Phusion polymerase | * 0.2 μl Phusion polymerase | ||
| − | * | + | * 5 μl H<sub>2</sub>O |
We dilute to Phusion to make the pipetting a bit easier and obtain the right final concentration for all ingredients. | We dilute to Phusion to make the pipetting a bit easier and obtain the right final concentration for all ingredients. | ||
| − | + | <BR> | |
Run using program (bart -> pombe -> colony PCR) | Run using program (bart -> pombe -> colony PCR) | ||
{| class="wikitable" | {| class="wikitable" | ||
| − | !Number of cycles | + | !Number of cycles |
| − | !Temperature | + | !Temperature |
!Duration | !Duration | ||
|- | |- | ||
| Line 38: | Line 38: | ||
|35X || 94°C || 30 sec | |35X || 94°C || 30 sec | ||
|- | |- | ||
| − | | | + | | || 55-60°C || 30 sec |
|- | |- | ||
| − | | | + | | || 72°C || 2 min 30 sec |
|- | |- | ||
|1X || 72°C || 7 min | |1X || 72°C || 7 min | ||
|- | |- | ||
| − | | | + | | || 7°C || Inf |
|} | |} | ||
Latest revision as of 21:42, 3 January 2021
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 |
