LacZ transgenic mouse genotyping protocol
This protocol is for checking of presence of LacZ gene (beta galactosidase reporter gene) in transgenic mice. The primers below recognize sequences within the beta galactosidase gene.
Mouse tail DNA preparation: see Mouse Tail DNA Extraction Protocol
1. Primer sequences:
(1). LacZ primer pair:
Forward primer: 5’-TTC ACT GGC CGT CGT TTT ACA ACG TCG TGA-3'
Reverse primer: 5’-ATG TGA GCG AGT AAC AAC CCG TCG GAT TCT-3’
PCR product size: 364 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-------50 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.
