Difference between revisions of "PCR Amplification of DNA"
From Bridges Lab Protocols
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*Run PCR Program. Normally use touchdown PCR ('''DAVETD''') as follows: | *Run PCR Program. Normally use touchdown PCR ('''DAVETD''') as follows: | ||
| − | #1 min at 94 | + | :#1 min at 94 |
| − | #30s at 65 | + | :#30s at 65 |
| − | #2 min/kb at 72 | + | :#2 min/kb at 72 |
| − | #30s at 94 | + | :#30s at 94 |
| − | #30s at 63 then -0.5/cycle | + | :#30s at 63 then -0.5/cycle |
| − | #2 min/kb at 72 | + | :#2 min/kb at 72 |
| − | #Repeat steps 4-6 28 times | + | :#Repeat steps 4-6 28 times |
| − | #30s at 94 | + | :#30s at 94 |
| − | #30s at 45 | + | :#30s at 45 |
| − | #11 min at 72 | + | :#11 min at 72 |
| − | # | + | :#Hold at 4 until ready |
*Purify PCR product if necessary using Qiagen kit (Add 5x PB) | *Purify PCR product if necessary using Qiagen kit (Add 5x PB) | ||
Revision as of 13:24, 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
- 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 min at 94
- 30s at 65
- 2 min/kb at 72
- 30s at 94
- 30s at 63 then -0.5/cycle
- 2 min/kb at 72
- Repeat steps 4-6 28 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)
