Stock No: 013144
Protocol 28825: Standard PCR Assay - Aim2<Gt(CSG445)Byg>
Version 2.2

Notes

This assay does not work well without the use of a HotstartTaq polymerase.  This assay cannot distinguish heterozygous from homozygous KO mice.

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 = ~200 bp

Heterozygote = ~200 bp and 487 bp

Wild type = 487 bp

A duplication of part of the Aim2 genomic region at the 3' end of the integration site makes this assay unable to distinguish heterozygous from homozygous KO mice by PCR.

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
12906 GTT TGG CTC AGA AAT GTC CAG Common A
12907 CCC ACA TAA CCT GGG ATT AGT T Wild type Reverse A
12908 AAG GGT CTT TGA GCA CCA GA Mutant Reverse A

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
12906 0.50 uM
12907 0.50 uM
12908 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.