Stock No: 001162
Protocol 14328: Pyrosequencing Assay - H2-Ab1<bm12>
Version 2.0

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

Sequence

CGTGGTCCCCGCTGTCCCCCGGGCTGACCGCGTCCGTCCGCAGGGCATTTCGTGTACCAGTTCATGGGCG
AGTGCTACTTCACCAACGGGACGCAGCGCATACGATATGTGACCAGATACATCTACAACCGGGAGGAGTA
CGTGCGCTACGACAGCGACGTGGGCGAGCACCGCGCGGTGACCGAGCTGGGGCGGCCAGACGCCGAGTAC
TGGAACAGCCAGCCGGAG(a/t)TCCTGGAGC(g/a)AA(c/a)GCGGGCCGAGCTGGACACGGTGTGCAGACACAACTACG
AGGGGCCGGAGACCCACACCTCCCTGCGGCGGCTTGGTGAGCGCGGCGGGTCCCGCGGGAGCGCGGCGGG
CCGTGAGGGGACGCGGAGCAGAGTTCCCGCCTGAGGAGCTGCATGGCCTCCTTCCTCCCGTCTGCCCTGC
ACCACCTAGCGCCTCCTTGGAGTCTCCCTCCTGTCTCACCTCTGCCCTCTGCCCTTTGCCTCATGAGGCC

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
11402 Fluorophore AGA CGC CGA GTA CTG GAA CAG C
11403 Fluorophore CGG CCC CTC GTA GTT GTG
11404 Fluorophore AAC AGC CAG CCG GAG

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.00
MgCl2 2.00
dNTPS-kapa 0.20
11402 0.50
11403 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.