Genotyping Protocols Database
Stock No: 002667
Protocol 29270: Standard PCR Assay - Cd28<tm1Mak>alternate1
Version 3.2

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 = 524 bp
Heterozygote = 327 bp and 524 bp
Wild type = 327 bp

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
13967 CTT TGA TTT CAG GGC AAT GG Wild type Forward A
13968 TTG ACG TGC AGA TTC CAG AG Common A
oIMR9692 CCA GTC ATA GCC GAA TAG CC Mutant Forward A

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
13967 0.50 uM
13968 0.50 uM
oIMR9692 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
002666 B6.129S2-Cd28tm1Mak/J
002667 C.129S2(B6)-Cd28tm1Mak/J
004761 NOD.129S2(B6)-Cd28tm1Mak/JbsJ
3 strains use this protocol