Q5 PCR
Updated 12/23/2014 08:48 AM
Introduction
Materials
- 50µL Reaction
- 0.5µL Q5 DNA polymerase
- 2.5µL 10µM forward primer
- 2.5µL 10µM reverse primer
- 10µL 5x Q5 reaction buffer
- 0.5µL template DNA
- 1µL dNTP mix (10mM each)
- 33µL ddH2O
Procedure
Setup
- Mix everything together
Master Mix
Table1
A A | B B | C C | |
1 1 | reagent | concentration | pipet this much |
2 2 | forward primer (µM) | 10 | 2.5 |
3 3 | reverse primer (µM) | 10 | 2.5 |
4 4 | 5x Q5 rxn buffer | 10 | |
5 5 | template DNA | doesn't matter! | 0.5 |
6 6 | dNTP mix (mM each) | 10 | 1 |
7 7 | Q5 polymerase | 0.5 | |
8 8 | water | "=B10*B11-sum(C2:C7)" --> | 33 |
9 9 | "=(B10*B11)-sum(C2:C7)" --> | 33 | |
10 10 | how many reactions are you doing? | 2 | |
11 11 | how many µL do you want each reaction to be? | 25 |
Thermal cycler program
- 98C for 5min
- 98C for 10s
- 55C for 30s
- 72C for 20s/kb
minimum time 30s
- Repeat steps 2-4 (3-5?) 29 times
- 72C for 10min
- 10C hold
PCR amplification using Q5 (regular or hotstart) DNA polymerase from NEB. The error rate is comparable to Phusion, but anecdotally, some products seem to amplify better using Q5 vs Phusion.