1.primer should contain 15-18 genspecific bases,
2.add restriction site to 5Õ end (if tagging a gene,make sure sequence is in frame with tag, add bases if necessary).
3.Add 5Õ overhang to restriction site to make sure enzyme is cutting properly (number of bases added varies with enzyme used, see Reagents binder, Restriction enzymes)
4.GC-content should be 40-60%.
5. Annealing temperature of both oligos should be roughly the same, typically ~60¡C Tm (Annealing temperatures of 50-72¡C, depends also on polymerase)
Order primers online from:
IDT: order standard desalting, 25nmol
Or Invitrogen: desalted, 25nmol
dissolve oligos at 100µM in H2O (use extra clean water) for 10min at 65¡C
dilute an aliquot of oligo solution to 10µM for use in PCR
store primers at -20¡C, put info sheet in common lab binder
Template: dilute DNA to 20ng/µl
use extra clean water, filter tips and tubes (drawer ÒPCR tubesÓ), wear gloves!!
Use Proofreading Polymerase
e.g. Pfu Ultra II Fusion HS DNA Polymerase (Stratagene)
dNPTs from Wisent (100mM total stock), aliquoted as 10mM working solutions.
PCR reaction: 50µl
Prepare on ice
Template 1µl = 20ng (5-30ng)
10xPfu Ultra II Buffer 5µl
Primer 5Õ 10µM 1µl
Primer 3Õ 10µM 1µl
dNTP (10mM total) 5µl
Pfu Ultra II Polymerase 1µl
Primer Tm - 5¡C 20sec
72¡C 15sec/kb 30cycles
check 4µl on gel
Anne Schohl, 2008