Protocol 14262: Pyrosequencing Assay - Kit<W-41J>
Version 3.0

Notes

This genotyping assay uses pyrosequencing technology and is run on the Biotage PSQ 96MA. The Jackson Laboratory is not posting the complete details of our pyrosequencing genotyping assays as the primers for pyrosequencing cannot be used for sequencing using more traditional methods. The wild type and mutant nucleotides and the flanking DNA sequence are provided below.
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 = A/A
Heterozygote = A/G
Wild type = G/G

Sequence

CAAAGCAGTCTGGGAAATGTAGTTATAGTTGGGAGAGGCCATATATGTTGTTGACAAA
AAAAAAAAAAAAAAAAGAAGAAGAAGAAGAAAGTTGCTAGTTGAGGCCATTACTAG
AAGGAAGGTTAGAACCCCTGGACTTCTCTGCTCTTAGTTTACTGTCCTATACTGACTCA
ACACCCCTATTTTAAAGGGAGATATTAGAATTTTGAATTATAAGTAGGGGAGGTGGCTG
GAGGTCACAAGGTTTAAGGTCCTCGTCTATCGCTGTCTTCATTAGCTGCTTGAATTTGC
TGTGTTCCGTTCTAGGCACGACTGCCC(g/a)TGAAGTGGATGGCACCAGAGAGCATTTT
CAGCTGCGTGTACACATTTGAAAGTGATGTCTGGTCCTATGGGATTTTCCTCTGGGAG
CTCTTCTCCTTAGGTAAAATGTTCCTTACCCAAGATGCCCTCACTGTCTAAAAGCTTCT
GTCGTCTCCCGCCCACATGGACACTGATTTGCCACCTCTCTGCACCCAGGAAGCAGCC
CCTACCCAGGGATGCCGGTCGACTCCAAGTTCTACAAGATGATCAAGGAAGGCTTCCG
GATGGTCAGCCCGGAGCACGCGCCTGCC

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
poIMR0124 TTG CTG TGT TCC GTT CTA GGC Forward
poIMR0125 Fluorophore GCT CTC TGG TGC CAT CCA CT Reverse
poIMR0126 CGT TCT AGG CAC GAC TG SEQ

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.00
MgCl2 2.00
dNTPS-kapa 0.20
poIMR0124 0.50
poIMR0125 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

Stock Number Strain Name
006564 B6(C)-KitW-41J Gusbmps/BrkJ
026622 NOD.Cg-KitW-41J Tyr + Prkdcscid Il2rgtm1Wjl/ThomJ
2 strains use this protocol