PCR Amplification for Cloning

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 T_m - 5°C 20sec

72°C 15sec/kb 30cycles

72°C 5min

check 4µl on gel

use everything for restriction digest (except if band on gel is extremely strong,use 30µl)

Anne Schohl, 2008