(1). BAF250A genotyping primer pair:
Forward primer: 5’-GTA ATG GGA AAG CGA CTA CTG GAG-3’
Reverse primer1: 5’-TGT TCA TTT TTG TGG CGG GAG-3’
PCR product size:
Homozygous BAF250A-flox allele: 812 bp
Wild-type allele: 632 bp
Knockout allele: 268 bp
(2). BAF250A-Flox-specific primer pair:
Forward primer: 5'-GTG TTG CCG CGG ATA ACT TC-3'
Reverse primer: 5'-TAC AGG CGC GCC ATA ACT TC-3'
PCR Product size: 601 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 5 minutes
2). PCR cycles:
94°C-------30 sec
59°C-------25 sec
72°C-------50 sec
For 35-40 cycles
3). Extension: 72°C for 10 minutes
4). Store at 4°C.
3. Runing agrose gel
