View source for PCR Amplification of DNA (KOD Polymerase)

==Materials==
*Primers – order from IDT prediluted to 100 mM in TE.  Make working solution of 1 uM (100x) in water with both sense and antisense primers combined (300uL water, 3uL of each primer)
*dNTPs – dilute to 2 mM each in water, make single use aliquots (50 uL aliquots... 10uL of each dNTP, 460uL water)
*Template – generally 1uL or less of a plasmid miniprep
*KOD Polymerase

==Protocol==
*Use the following volumes per reaction
::*KOD Buffer #1, 5 uL of 10X buffer (Dave's Molecular Biology Stuff Box)
::*Primers, 10uL of 1uM stock solution in water (both primers combined)
::*dNTPs, 5uL of 2 mM (Dave's Molecular Biology Stuff Box)
::*MgCl, 3uL of 3 mM (Dave's Molecular Biology Stuff Box)
::*Sterile water, 28 uL
::*Template 1 uL of a miniprep
::*Polymerase 0.4 uL (KOD Polymerase found in "Enzymes" box in freezer)

*Run PCR Program (approx 3.5 to 4 hours).  Normally use touchdown PCR ('''TD-KOD''') as follows:
:#15s at 94
:#5s at 65
:#30s at 72
:#15s at 94
:#5s at 63 then -0.5/cycle
:#30s at 72
:#Repeat steps 4-6 30 times
:#30s at 94
:#30s at 45
:#11 min at 72
:#Hold at 4 until ready
*Purify PCR product if necessary using Qiagen kit (Add 5x PB)