Difference between revisions of "PCR Amplification of DNA"

Revision as of 13:08, 5 May 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
• dNTPs – dilute to 2 mM each in water, make single use aliquots (50 uL)
• Template – generally 1uL or less of a plasmid miniprep
• Polymerase – use Pfu Turbo for cloning and Taq for noncloning. Use appropriate buffer.

Protocol

1. Use the following volumes per reaction

• Buffer, 5 uL of 10X buffer
• Primers, 10uL of 1uM stock solution in water (both primers combined)
• dNTPs, 5uL of 2 mM
• Sterile water, 28 uL
• Template 1 uL

Polymerase 1 uL

• Run PCR Program. Normally use touchdown PCR (DAVETD) as follows:

1. 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)