Protocol 29723: Standard PCR Assay - Gt(ROSA)26Sortm1(birA)Mejr
Version 3.2

Notes

This assay does not work well without the use of a Hotstart Taq polymerase.
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 = ~500 bp and 367 bp
Wild type = 367 bp

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
9414 TTC AGA CAC TGC GTG ACT A
9415 GGC TCC AAT GAC TAT TTG C A
9416 GTG TAA CTG TGG ACA GAG GAG A
9417 GAA CTT GAT GTG TAG ACC AGG A

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
9414 0.50 uM
9415 0.50 uM
9416 0.50 uM
9417 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
010920 FVB;129P2-Gt(ROSA)26Sortm1.1(birA)Mejr/J
018121 STOCK Gt(ROSA)26Sortm1(birA)Mejr Gata4tm3.1Wtp/J
2 strains use this protocol