Vhl knockout mouse genotyping protocol
This protocol is for Vhl knockout transgenic mouse genotyping from Jackson Laboratory. The strain number is C;129S-Vhltm1Jae/J. Stock number is 004081.
Mouse tail DNA preparation: see Mouse Tail DNA Extraction Protocol
1. Primer sequences (from The Jackson Laboratory):
Vhl primer pair:
Forward primer: 5’-CTC AGG TCA TCT TCT GCA ACC-3'
Reverse primer: 5’-TCT GTC TTG GCC TCC TGA GT-3’
Expected PCR products:
Mutant =~1150 bp
Heterozygote = ~1150 bp and 933 bp
Wild type =933 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-------80 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.
Redesigned Vhl genotypng primers by Jindong Chen:
Forward primer: 5'-GGC AGA GTG TGG GTG CGT CC-3'
Reverse primer: 5'-ACA CCT GAG AGC GGC TTC ACT-3'
Expected PCR products:
Mutant =509 bp
Heterozygote = 509 bp and 311 bp
Wild type =311 bp
Redesigned Vhl-flox-specific primer pair (if the above primers works very well, this primer pair is not needed):
Forward primer: 5'-GGC AGA GTG TGG GTG CGT CC-3'
Reverse primer: 5'-CGT GCG GCC TCG ACG GTA TC-3'
Expected PCR product: Mutant=242 bp
Use the same PCR reaction condition above to run PCR.
Redesigned Vhl-wt-specific primer pair (for confirmation):
Forward primer: 5'-CAA GCC TGA CCA CGT AAG CTT A-3'
Forward primer-New1: 5'-CAA GCC TGA CCA CGT AAG CTT ATT-3'
Reverse primer: 5'-TCT GTC TTG GCC TCC TGA GTC-3'
Expected PCR product: Wild Type=317 bp
Note: other primers (not confirmed)
Vhl-WTF: 5'-ACC AGA AGA GTG CCA GAT CC-3'
Vhl-WTR: 5'-CTC ACA ACC ATA TGG GAC CC-3'
Vhl-748-R: 5'-GTG TTA TTT GGT TGT AAC AAA-3'
