(1). Sglt2-Cre primer pair1:
Forward primer: 5’-GGT AAG GAG GTG CAG TCT GG-3’
Reverse primer: 5’-AGG CAA ATT TTG GTG TAC GG-3’
PCR product size: 285 bp
(2). Sglt2-Cre primer pair2:
Forward primer: 5’-CCT GAA ACA AGG CTG AGG AA-3’
Reverse primer: 5’-CAG GTT CTT GCG AAC CTC AT-3’
PCR product size: 284 bp
(3). Sglt2-Cre primer pair3:
Forward primer: 5’-GCC TGG GTT GTT CTG AGT GT-3’
Reverse primer: 5’-GGC AAA CGG ACA GAA GCA TT-3’
PCR product size: 431 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
