Protocol 27509: Standard PCR Assay - Tnfrsf1a<tm1Imx>-Alternate 3
Version 1.2

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 = ~270 bp
Heterozygote = ~270 bp and 362 bp
Wild type = 362 bp

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
12834 GCT ACT TCC ATT TGT CAC GTC C Mutant Reverse A PGK R
22431 ATG GGG ATA CAT CCA TCA GG Common A
22432 GGG GAA CAT CAG AAA CAA GC Wild type Reverse A

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
12834 0.50 uM
22431 0.50 uM
22432 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
003243 B6.129S-Tnfrsf1atm1Imx Tnfrsf1btm1Imx/J
003244 B6;129S-Tnfrsf1atm1Imx Il1r1tm1Imx/J
003242 C57BL/6-Tnfrsf1atm1Imx/J
024314 NOD.Cg-Tnfrsf1btm1Imx Tnfrsf1atm1Imx/J
4 strains use this protocol