Protocol 25492: Standard PCR Assay - Tg(Thy1-TARDBP)4Singh
Version 2.2

Notes

Donating Investigator reports this transgene had integrated at locus 6qB3 of the mouse genome (nucleotide 56,524,796) and did not interrupt any known gene.

WT F and Common R primers amplify a PCR product from mouse of 303 bp.  Tg F primer is specific for the Tg.

This assay will distinguish hemizygous from homozygous transgenic animals.

Changed common from .13791 (no amplification with Tg)  to .13790 per Kai (successful amplification with Tg)  SC 3-1-12.

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 = ~500 bp
Heterozygote = 303 bp and ~500 bp
Wild type = 303 bp

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
13790 TGA AAT CCG GGT GGT ATT GG Common A
13791 GGT GAG TTT AAC CTT CAA GGG CT Wild type A
13792 AGC TTG CTA GCG GAT CCA GAC Transgene A

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
13790 0.50 uM
13791 0.50 uM
13792 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.