Stock No: 003090
Protocol 28818: Standard PCR Assay - Generic Cd4
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

HET = 381 bp and 225 bp
MUT= 225 bp
WT= 381 bp
 

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
oIMR0099 CCT CTT GGT TAA TGG GGG AT Wild type Forward A
oIMR0100 TTT TTC TGG TCC AGG GTC AC Wild type Reverse A
oIMR5241 GTG TTG GGT CGT TTG TTC G Mutant Forward A PGK-neo-pA

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
oIMR0099 0.50 uM
oIMR0100 0.50 uM
oIMR5241 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
002663 B6.129S2-Cd4tm1Mak/J
003090 NOD.129S6(B6)-Cd4tm1Knw/DvsJ
2 strains use this protocol