Stock No: 003726
Protocol 26183: Standard PCR Assay - Cd14<tm1Frm>
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 = ~600 bp
Heterozygote = ~600 bp and 840 bp
Wild type = 840 bp
Separated by gel electrophoresis on a 1.5% agarose gel.

Sequence

Validation/LC Conversion data attached to rec 129912

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
oIMR0662 CCG CTT CCA TTG CTC AGC GG Mutant Forward A
oIMR1314 CCA AGT TTT AGC GCT GCG TAA C Wild type Forward A
oIMR1315 GCC AGC CAA GGA TAC ATA GCC Common A

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
oIMR0662 0.50 uM
oIMR1314 0.50 uM
oIMR1315 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.