PCR Amplification of DNA: Difference between revisions
Created page with '==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 – dil...' |
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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 | ||
*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 | ||
*Sterile water, 28 uL | ::*Sterile water, 28 uL | ||
*Template 1 uL | ::*Template 1 uL | ||
::Polymerase 1 uL | |||
*Run PCR Program. Normally use touchdown PCR ('''DAVETD''') as follows: | |||
##1 min at 94 | ##1 min at 94 | ||
##30s at 65 | ##30s at 65 | ||
| Line 26: | Line 26: | ||
##11 min at 72 | ##11 min at 72 | ||
##hold at 4 until ready | ##hold at 4 until ready | ||
*Purify PCR product if necessary using Qiagen kit (Add 5x PB) | |||