(1). BAF180-floxed primer pair:
Forward primer: 5’-TAC AAT GAT GCC CAT ATC CTG GAG-3’
Reverse primer1: 5’-GGT CTC AAA GTC AGG GTC ACA AGG-3’
Reverse primer2: 5'-CTG CAA CTC TTT GTC CTT ACA CGA TG-3'
PCR product size:
Homozygous BAF180-flox allele: 557 bp
Wild-type allele: 431 bp
Heterozygotes: 557bp +431 bp
(2). BAF180-knockout primer pair:
Forward primer: 5’-CCG GCT CCG CGG ATA ACT TCG TAT-3’
Reverse primer2: 5’-CTG CAA CTC TTT GTC CTT ACA CGA TG-3’
PCR product size:
Knockout allele: 336 bp
Flox band: 893 bp
No Wild type band.
(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:
95°C-------30 sec
59.5°C-------20 sec
72°C-------45 sec
For 5 cycles
then
94°C-----30 sec
59.5°C-------20 sec
72°C-------45 sec
For 35 cycles
3). Extension: 72°C for 10 minutes
4). Store at 4°C.
3. Runing agrose gel
