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 ~60C Tm (Annealing temperatures of 50-72C, depends also on polymerase)

 

Order primers online from:

 IDT: order standard desalting, 25nmol

Or Invitrogen: desalted, 25nmol

dissolve oligos at 100M in H2O (use extra clean water) for 10min at 65C

 

dilute an aliquot of oligo solution to 10M for use in PCR

store primers at -20C, put info sheet in common lab binder

Template: dilute DNA to 20ng/l

 

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: 50l

Prepare on ice

 

Template                               1l = 20ng (5-30ng)

10xPfu Ultra II Buffer           5l

Primer 5 10M                    1l

Primer 3 10M                    1l

dNTP (10mM total)              5l

H2O                                         36l

Pfu Ultra II Polymerase      1l

 

95C                           2min

95C                           20sec

Primer Tm - 5C        20sec

72C                           15sec/kb       30cycles

72C                           5min

 

check 4l on gel     

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

 

Anne Schohl, 2008