Primer design:
1.primer should contain
15-18 genspecific bases,
2.add restriction site to 5’
end (if tagging a gene,make sure sequence is in frame with tag, add bases if
necessary).
3.Add 5’ 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 “PCR tubes”), 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 5’ 10µM 1µl
Primer 3’ 10µM 1µl
dNTP (10mM total) 5µl
H2O 36µl
Pfu Ultra II Polymerase 1µl
95°C 2min
95°C 20sec
Primer Tm - 5°C 20sec
72°C 15sec/kb
30cycles
72°C 5min
check 4µl on gel
Anne Schohl, 2008