Difference between revisions of "PCR Amplification of DNA"
From Bridges Lab Protocols
Davebridges (Talk | contribs) m (→Protocol) |
|||
| Line 1: | Line 1: | ||
==Materials== | ==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 | + | *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) | + | *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. | *Polymerase – use Pfu Turbo for cloning and Taq for noncloning. Use appropriate buffer. | ||
| Line 7: | Line 7: | ||
==Protocol== | ==Protocol== | ||
*Use the following volumes per reaction | *Use the following volumes per reaction | ||
| − | ::*Buffer, 5 uL of 10X buffer | + | ::*Buffer, 5 uL of 10X buffer (Dave's fridge) |
::*Primers, 10uL of 1uM stock solution in water (both primers combined) | ::*Primers, 10uL of 1uM stock solution in water (both primers combined) | ||
| − | ::*dNTPs, 5uL of 2 mM | + | ::*dNTPs, 5uL of 2 mM ("molecular biology stuff" box in freezer) |
::*Sterile water, 28 uL | ::*Sterile water, 28 uL | ||
::*Template 1 uL | ::*Template 1 uL | ||
| − | ::*Polymerase 1 uL | + | ::*Polymerase 1 uL (turbo pfu found in "enzymes" box in freezer) |
| − | *Run PCR Program. Normally use touchdown PCR ('''DAVETD''') as follows: | + | *Run PCR Program (approx 3.5 to 4 hours). Normally use touchdown PCR ('''DAVETD''') as follows: |
:#1 min at 94 | :#1 min at 94 | ||
:#30s at 65 | :#30s at 65 | ||
Revision as of 16:02, 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 (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
- Buffer, 5 uL of 10X buffer (Dave's fridge)
- Primers, 10uL of 1uM stock solution in water (both primers combined)
- dNTPs, 5uL of 2 mM ("molecular biology stuff" box in freezer)
- Sterile water, 28 uL
- Template 1 uL
- Polymerase 1 uL (turbo pfu found in "enzymes" box in freezer)
- Run PCR Program (approx 3.5 to 4 hours). 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)
