FLPer transgenic mouse genotyping protocol
This protocol is for FLPer transgenic mouse genotyping to check whether the mice under examination carry the FLP gene which would remove any marker gene for selection.
Mouse tail DNA preparation: see Mouse Tail DNA Extraction Protocol
1. Primer sequences:
(1). FLPer primer pair:
Forward primer: 5’-CAC TGA TAT TGT AAG TAG TTT GC-3'
Reverse primer: 5’-CTA GTG CGA AGT AGT GAT CAG G-3’
PCR product size: 725 bp
2. PCR Reaction
(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-------60 sec
For 35-40 cycles
3). Extension: 72°C for 10 minutes
4). Store at 4°C.
3. Runing agrose gel
Prepare 2% agrose gel in TAE or TBE buffer. DNA marker: 100 bp ladder.
