Stock No: 012252
Protocol 14834: Pyrosequencing Assay - Tbce<pmn>
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

Sequence

TAAATGGAGGCAGCCTTTGACTTCACACCTCACCTGCCCTTGGAGGGTGGCATG
GATAGCATGCACCATCAGATCGGTCTTACTGCTCCCTACTTGATTTTAATTTTAGAT
GCCGGGCAGAGAAATTGAACTAGAAAATGACCTACAGCCATTACAGTTTTATTCTG
TGGAAAATGGAGATTGCCTACTAGTGCGA(t/g)GGTAACAGTCCAACCGTAAGAGTTT
AACGACTGGAATGTATACACCAGAAACAAAACCTTTGCCCTCACAAATGATGTTAT
GTTTTGTTGGGAAAAGACCATTTGGTTGTATATTAATAGAATAATAAAGGCTTTGAT
TACTATTGATTTCTCTGCCCTTTCTGTTTTCACACCTTACTGCATTCACTATGTGAA

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
8978 TGT GGA AAA TGG AGA TTG CCT ACT
8979 Fluorophore CGT TAA ACT CTT ACG GTT GGA CTG
8980 GGA GAT TGC CTA CTA GTG C

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.00
MgCl2 2.00
dNTPS-kapa 0.20
8978 0.50
8979 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.