Stock No: 002252
Protocol 26241: Standard PCR Assay - Il2<tm1Hor>
Version 2.2

Notes

Validation/LC Conversion data attached to rec 165561

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

Evagreen

HT-TD65-60  cycling                      RS Macro                   Genescanning analysis

DO NOT USE TM Calling analysis, cannot differentiate between Het/Hom

Mutant = 500 bp
Heterozygote = 324 bp and 500 bp
Wild type = 324 bp

 

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
oIMR0041 TCG AAT TCG CCA ATG ACA AGA CGC T A
oIMR7338 CTA GGC CAC AGA ATT GAA AGA TCT A
oIMR7339 GTA GGT GGA AAT TCT AGC ATC ATC C A

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
oIMR0041 0.50 uM
oIMR7338 0.50 uM
oIMR7339 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
002252 B6.129P2-Il2tm1Hor/J
002229 C.129P2(B6)-Il2tm1Hor/J
002228 C3.129P2(B6)-Il2tm1Hor/J
002573 NOD.129P2(B6)-Il2tm1Hor/DvsJ
4 strains use this protocol