Genotyping Protocols Database
Stock No: 002692
Protocol 29750: Standard PCR Assay - Il12a<tm1Jm>
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 = 172 bp
Heterozygote = 172 bp and 650 bp
Wild type = 650 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
oIMR0158 CTG AAT GAA CTG CAG GAC GA A
oIMR0159 ATA CTT TCT CGG CAG GAG CA A
oIMR0459 CAG CAT GTG TCA ATC ACG A
oIMR0460 TCA CCA TGT CAT CTG TGG A

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
oIMR0158 0.50 uM
oIMR0159 0.50 uM
oIMR0459 0.50 uM
oIMR0460 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
002691 C.129S1(B6)-Il12atm1Jm/J
002692 B6.129S1-Il12atm1Jm/J
2 strains use this protocol