Genotyping Protocols Database
Stock No: 004432
Protocol 22327: Standard PCR Assay - Tbx21<tm1Glm>
Version 1.3

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 = ~450 bp
Heterozygote = 263 bp and ~450 bp
Wild type = 263bp
Separated by gel electrophoresis on a 1.5% agarose gel.

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
oIMR1717 GCG CGA AGG GGC CAC CAA AGA ACG GAG Mutant A
oIMR1718 GAC TGA AGC CCC GAC CCC CAC TCC TAA G Wild type A
oIMR1719 TGG GCA TAC AGG AGG CAG CAA CAA ATA Common A

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
oIMR1717 0.50 uM
oIMR1718 0.50 uM
oIMR1719 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
004648 B6.129S6-Tbx21tm1Glm/J
004432 C.129S6-Tbx21tm1Glm/J
2 strains use this protocol