Bridges Lab Protocols

PCR Amplification of DNA (KOD Polymerase): Difference between revisions

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*dNTPs – dilute to 2 mM each in water, make single use aliquots (50 uL aliquots... 10uL of each dNTP, 460uL water)
*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
*Template – generally 1uL or less of a plasmid miniprep
*Polymerase – use Pfu Turbo for cloning and Taq for noncloning.  Use appropriate buffer.
*KOD Polymerase


==Protocol==
==Protocol==
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*Run PCR Program (approx 3.5 to 4 hours).  Normally use touchdown PCR ('''TD-KOD''') as follows:
*Run PCR Program (approx 3.5 to 4 hours).  Normally use touchdown PCR ('''TD-KOD''') as follows:
:#1 min at 94
:#15s at 94
:#30s at 65
:#5s at 65
:#2 min/kb at 72
:#30s at 72
:#30s at 94
:#15s at 94
:#30s at 63 then -0.5/cycle
:#5s at 63 then -0.5/cycle
:#2 min/kb at 72
:#30s at 72
:#Repeat steps 4-6 28 times
:#Repeat steps 4-6 30 times
:#30s at 94
:#30s at 94
:#30s at 45
:#30s at 45
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