Appending Prefix and Suffix

Assembling Reaction

1. Get PCR tube (0.6mL tubes), not the strip tubes
2. Thaw Pfx supermix on ice
3. Get primers for gene of interest

1. Add the following:

Calculating reaction conditions

1. Use idtdna.com or VectorNTI to calculate primer temperatures of primers without common overhangs (Base pairs 30 to end when read 5' to 3')

1. Pfx elongates at a rate of 500bp per 30s. Look up the length of the gene of interest and calculate time of elongation.
2. If you get the melting temperature of your primer from genious, the annealing temperature will be that number subtracted by 2.

Programming a thermocycler

..... need to look up what kind we have in TK's lab

A typical cycle follows:

Initial Denaturation: 5min @ 95C

Loop (30-35 cycles) 
Denaturation: 30s @ 95C 
Annealing: 30s @ temperature calculated above (55-65C normally) 
Elongation: 30s per every 500 base pairs @ 68C

Final Elongation: 15min at 68C 
Store: 16C