Stock No: 007020
Protocol 22989: Standard PCR Assay - Tlr1<tm1Flv>
Version 1.3

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 = 282 bp
Heterozygote = 282 bp and 537 bp
Wild type = 537 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
oIMR6966 GCC AAA CGC AAA CCT TAC CAG AGT G Wild type Forward A
oIMR6967 ACG GAC ACA TCC AGA AGA AAA CGG Wild type Reverse A
oIMR6968 TTC GGC TAT GAC TGG GCA CAA CAG Mutant Forward A Neo
oIMR6969 TAC TTT CTC GGC AGG AGC AAG GTG Mutant Reverse A Neo

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
oIMR6966 0.50 uM
oIMR6967 0.50 uM
oIMR6968 0.50 uM
oIMR6969 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

This is the only strain that uses this protocol.