PCR Amplification of DNA: Difference between revisions
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==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. | ||
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==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 | ||