Stock No: 007806
Protocol 23025: Standard PCR Assay - Stat5a<tm1Mam>
Version 2.3

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 = 168 bp
Heterozygote = 168 bp and 447 bp
Wild type = 447 bp
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
oIMR7570 GGA GAC TCA TCT AGA CTT TGA TGC TTA TAC Wild type Forward A
oIMR7571 AAG ATC TAA CAG GTC TGA CTT AGA AAG AGG Wild type Reverse A
oIMR7572 CAG ACT GCC TTG GGA AAA GT Mutant Forward A
oIMR7573 CGG CGA GTC TGA TTC TCT TC 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
oIMR7570 0.50 uM
oIMR7571 0.50 uM
oIMR7572 0.50 uM
oIMR7573 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
002833 B6;129S6-Stat5atm1Mam/J
007806 C.129S(B6)-Stat5atm1Mam/J
003502 FVB.129S6-Stat5atm1Mam/J
3 strains use this protocol