"
Team:Washington/Protocols/PCR
From 2011.igem.org
(Difference between revisions)
m |
(→General PCR Protocol) |
||
| Line 8: | Line 8: | ||
##1uL 25mM dNTP's | ##1uL 25mM dNTP's | ||
##10uL Phusion HF Buffer | ##10uL Phusion HF Buffer | ||
| - | ##0.5uL Forward Primer (Tm | + | ##0.5uL Forward Primer (Tm XX <sup>o</sup>C)* |
| - | ##0.5uL Reverse Primer (Tm | + | ##0.5uL Reverse Primer (Tm YY <sup>o</sup>C)* |
##0.5uL Phusion polymerase | ##0.5uL Phusion polymerase | ||
##36.5uL diH2O | ##36.5uL diH2O | ||
#Amplification PCR Reaction | #Amplification PCR Reaction | ||
| - | ## | + | ##98 <sup>o</sup>C - 30s |
| - | ## | + | ##98 <sup>o</sup>C - 10s |
| - | ## | + | ##ZZ <sup>o</sup>C - 10s* |
| - | ## | + | ##72 <sup>o</sup>C - 30s/kb target gene |
##Repeat 2-4 29x | ##Repeat 2-4 29x | ||
| - | ## | + | ##72 <sup>o</sup>C - 5min |
| - | ## | + | ##10 <sup>o</sup>C - forever |
| + | * Tms of all primers must first be calculated after primer design. Annealing temperature (represented by ZZ above) should be 2 <sup>o</sup>C higher than XX or YY, whichever is lower. | ||
Refer for further details to NEB's online protocol for Phusion: | Refer for further details to NEB's online protocol for Phusion: | ||
http://www.neb.com/nebecomm/products/protocol87.asp | http://www.neb.com/nebecomm/products/protocol87.asp | ||
Revision as of 02:35, 16 September 2011
General PCR Protocol
- Setup Amplification PCR Reaction
- 1uL Template
- 1uL 25mM dNTP's
- 10uL Phusion HF Buffer
- 0.5uL Forward Primer (Tm XX oC)*
- 0.5uL Reverse Primer (Tm YY oC)*
- 0.5uL Phusion polymerase
- 36.5uL diH2O
- Amplification PCR Reaction
- 98 oC - 30s
- 98 oC - 10s
- ZZ oC - 10s*
- 72 oC - 30s/kb target gene
- Repeat 2-4 29x
- 72 oC - 5min
- 10 oC - forever
- Tms of all primers must first be calculated after primer design. Annealing temperature (represented by ZZ above) should be 2 oC higher than XX or YY, whichever is lower.
Refer for further details to NEB's online protocol for Phusion:
http://www.neb.com/nebecomm/products/protocol87.asp
