915
edits
Changes
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...'
==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)
*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)
