Stock No: 006825
Protocol 23607: Standard PCR Assay - Fas<lpr>
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=217 bp
Heterozygote = 179 bp and 217 bp
Wild type=179 bp
<p>Separated by gel electrophoresis on a 3% agarose gel.</p>

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
oIMR1678 GTA AAT AAT TGT GCT TCG TCA G Common A
oIMR1679 TAG AAA GGT GCA CGG GTG TG Mutant A
oIMR1680 CAA ATC TAG GCA TTA ACA GTG Wild type A

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
oIMR1678 0.50 uM
oIMR1679 0.50 uM
oIMR1680 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
000482 B6.MRL-Faslpr/J
000480 C3.MRL-Faslpr/J
002455 MRL.Cg-B2mtm1Unc Faslpr
003896 MRL/MpJ Faslpr-Foxq1sa-J/J
006825 MRL/MpJ-Faslpr/2J
000485 MRL/MpJ-Faslpr/J
004519 NOD.MRL(C3)-Faslpr/DoiJ
004922 NOD.MRL-Faslpr/Dvs
8 strains use this protocol