Stock No: 007746
Protocol 13661: Pyrosequencing Assay - Il4ra<tm2Tch>
Version 3.0

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 = GA
Wild type = AG

Sequence

AGGCCCTCTTTCAGGCAGCCCAGCCCAGAGTGCACCTACTCTGGCTTGCACGCAGGTC
CCACTTGTCCTTGCAGACAATCCTGCCTACCGGAGTTTTAGTGACTGCTGTAGCCCGGC
CCCAAATCCTGGAGAGCTGGCTCCAGAGCAGCAGCAGGCTGATCATCTGGAAGAAGA
GGAGCCTCCAAGCCCGGCTGACCCCCATTCTTCAGGGCCACCAATGCAGCCAGTGGA
GAGCTGGGAGCAGATCCTTCACATGAGTGTCCTGCAGCATGGGGCAGCTGCTGGCTCC
ACCCCAGCCCCTGCCGGTGGCTACC(agga)GAGTTTGTGCAGGCAGTGAAGCAGGGTG
CCGCCCAGGATCCTGGGGTGCCTGGTGTCAGGCCTTCTGGAGACCCCGGTTACAAGG
CCTTCTCGAGCCTGCTCAGCAGCAATGGCATCCGCGGGGACACAGCAGCAGCGGGG
ACTGACGATGGGCATGGAGGCTACAAGCCCTTCCAGAATCCTGTTCCTAACCAGTCC
CCTAGCTCCGTGCCCTTATTTACTTTCGGACTAGACACGGAGCTGTCACCCAGTCCTC
TGAACTCAGACCCACCCAAAAGCCCCCCAGAATGCCTTGGTCTGGAGCTGGGGCTC
AAAGGAGGTGACTGGGTGAAGGCCCCTCCTCCTGCAGATCAGGTGCCCAAGCCCTT
TGGGGATGACCTGGGCTTTGGTATTGTGTACTCGTCCCTCACTTGCCACTTGTGTGGC
CACCTGAAGCAACACCACAGCCAGGAGGAAGGTGGCCAGAGCCCCATCGTTGCTAG
CCCTGGCTGTGGCTGCTGCTACGATGACAGATCACCATCCCTG

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
pIMR192 CCC TGC CGG TGG CTA
poIMR0190 CGG CTG ACC CCC ATT CTT
poIMR0191 Fluorophore GCC TTG TAA CCG GGG TCT CC

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.00
MgCl2 2.00
dNTPS-kapa 0.20
poIMR0190 0.50
poIMR0191 0.50
Glycerol 5.00
Kapa 2G HS taq polym 0.01
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
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.

Strains Using This Protocol

This is the only strain that uses this protocol.