Protocol 26917: Standard PCR Assay - Tg(Alb-cre)21Mgn-Alternate 1
Version 2.2

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 = ~390 bp
Heterozygote = ~390 bp and 351 bp
Wild type = 351 bp

This assay is capable of distinguishing hemi from hom.

A 3% gel is required to separate bands.  An artifact band of ~150 bp is frequently present from the mutant allele.

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
20239 TGC AAA CAT CAC ATG CAC AC Wild type Forward A mouse Chr 13
20240 TTG GCC CCT TAC CAT AAC TG Common A mouse Chr 13
oIMR5374 GAA GCA GAA GCT TAG GAA GAT GG Mutant Forward A Alb promoter

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
20239 0.50 uM
20240 0.50 uM
oIMR5374 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

Stock Number Strain Name
003574 B6.Cg-Speer6-ps1Tg(Alb-cre)21Mgn/J
018961 B6N.Cg-Speer6-ps1Tg(Alb-cre)21Mgn/J
2 strains use this protocol