(1). Ksp-Cre primer pair 1:
Forward primer: 5’-GGT GGC ATT TTC CAC TCC TG-3'
Reverse primer: 5’-GCG AAC CTC ATC ACT CGT TG-3’
PCR product size: ~300 bp
(2). Ksp-Cre primer pair 2:
Forward primer: 5’-AAG TGC TCA TTG GGC TGT GT-3'
Reverse primer: 5’-GCA TTG CTG TCA CTT GGT CG-3’
PCR product size: ~600 bp
(1). Recipe for 15 ul reaction volume
DNA----------------------------1-2 ul (around10- 25 ng )
10x PCR buffer------------------1.5 ul
50mM MgCl2-------------------0.6 ul
5 mM dNTPs-------------------1.5 ul
10 uM Forward primer----------0.5 ul
10 uM Reverse primer-----------0.5 ul
5 units/ul Taq polymerase--------0.1 to 0.2 ul
Add ddH2O to -----------------15 ul
(2). PCR reaction:
1). Denature DNA at 95°C for 7 minutes
2). PCR cycles:
94°C-------30 sec
58°C-------20 sec
72°C-------45 sec
For 35-40 cycles
3). Extension: 72°C for 10 minutes
4). Store at 4°C.
3. Runing agrose gel
