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

Revision as of 21:23, 3 November 2009

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
• Polymerase – use Pfu Turbo for cloning and Taq for noncloning. Use appropriate buffer.

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:

1. 1 min at 94
2. 30s at 65
3. 2 min/kb at 72
4. 30s at 94
5. 30s at 63 then -0.5/cycle
6. 2 min/kb at 72
7. Repeat steps 4-6 28 times
8. 30s at 94
9. 30s at 45
10. 11 min at 72
11. Hold at 4 until ready

• Purify PCR product if necessary using Qiagen kit (Add 5x PB)