Neo (neomycin-resistant gene) transgenic mouse genotyping protocol
This protocol is for checking of presence of neomycin-resistant gene in transgenic mice.
Mouse tail DNA preparation: see Mouse Tail DNA Extraction Protocol
1. Primer sequences:
(1). Neor primer pair:
Forward primer: 5’-AGG ATC TCC TGT CAT CTC ACC TTG CTC CTG-3'
Reverse primer: 5’-AAG AAC TCG TCA AGA AGG CGA TAG AAG GCG-3’
PCR product size: 492 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.
