Genotyping Protocols Database
Stock No: 007913
Protocol 26533: Standard PCR Assay - Gli1<tm3(cre/ERT2)Alj>alternate3
Version 3.2

Notes

The genotyping protocol(s) presented here have been optimized for reagents and conditions used by The Jackson Laboratory (JAX). To genotype animals, JAX recommends researchers validate the assay independently upon receipt of animals into their facility. Reaction cycling temperature and times may require additional optimization based on the specific genotyping reagents used.

Expected Results

Mutant = ~100 bp
Heterozygote = ~100 bp and 136 bp
Wild type = 136 bp

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
oIMR1084 GCG GTC TGG CAG TAA AAA CTA TC Transgene Forward A
oIMR1085 GTG AAA CAG CAT TGC TGT CAC TT Transgene Reverse A
oIMR7888 GGG ATC TGT GCC TGA AAC TG Wild type Forward A
oIMR7889 CTT GTG GTG GAG TCA TTG GA Wild type Reverse A

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
oIMR1084 0.50 uM
oIMR1085 0.50 uM
oIMR7888 0.50 uM
oIMR7889 0.50 uM
Glycerol 6.50 %
Dye 1.00 X
Kapa 2G HS taq polym 0.03 U/ul
DNA

Cycling

Step Temp °C Time Note
1 94.0 --
2 94.0 --
3 65.0 -- -0.5 C per cycle decrease
4 68.0 --
5 -- repeat steps 2-4 for 10 cycles (Touchdown)
6 94.0 --
7 60.0 --
8 72.0 --
9 -- repeat steps 6-8 for 28 cycles
10 72.0 --
11 10.0 -- hold
JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.
JAX uses a 'touchdown' cycling protocol and therefore has not calculated the optimal annealing temperature for each set of primers.

Strains Using This Protocol

This is the only strain that uses this protocol.