(1). Bhd-floxed primer pair:
Forward primer: 5’-CCA TCA GCT CCA CCA GAA CC-3’
Reverse primer: 5’-TCT GAC CCA CTG CAG CCA C-3’
PCR product size:
Homozygous BHD-flox allele: 251 bp
Wild-type allele: 391 bp
Heterozygotes: 251bp +391 bp (note: sometimes, the 391 bp does not show up, better to use wild-type primers below to confirm)
(2). Bhd-knockout primer pair:
Forward primer: 5’-CCA TCA GCT CCA CCA GAA CC-3’
Reverse primer: 5’-CCG GTA CCT TAC AAG CCG A-3’
PCR product size:
Knockout allele: 152 bp
Wild type allele: 1 kb (usually does not show up, so always use wild-type primers below to confirm)
(3). Bhd-wild type-specific primer pair:
Forward primer: 5’-CCC TAT ATG TGC TGC AGG G-3’
Reverse primer: 5’-CAG AGG GAC AGG CAT CAA-3’
PCR product size: 171 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
