Latest revision as of 23:16, 28 September 2011

General PCR Protocol

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:

@@ Line 4: / Line 4: @@
 =General PCR Protocol=
-##Setup Amplification PCR Reaction
+#Setup Amplification PCR Reaction
-###1uL Template
+##1uL Template
-###1uL 25mM dNTP's
+##1uL 25mM dNTP's
-###10uL Phusion HF Buffer
+##10uL Phusion HF Buffer
-###0.5uL Forward Primer (Tm 65)
+##0.5uL Forward Primer (Tm XX <sup>o</sup>C)*
-###0.5uL Reverse Primer (Tm 65)
+##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
-###98C - 30s
+##98 <sup>o</sup>C - 30s
-###98C - 10s
+##98 <sup>o</sup>C - 10s
-###63C - 10s
+##ZZ <sup>o</sup>C - 10s*
-###72C - 30s/kb target gene
+##72 <sup>o</sup>C - 30s/kb target gene
-###Repeat 2-4 29x
+##Repeat 2-4 29x
-###72C - 5min
+##72 <sup>o</sup>C - 5min
-###10C - forever
+##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:
+http://www.neb.com/nebecomm/products/protocol87.asp