Protocol 14270: Pyrosequencing Assay - Pign<m1Nisw>

129S1.B6-Pign^m1Nisw/J

Stock No: 013562

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

WT=T/T
Mut=A/A

Sequence

ATGACATCCGTAGGGCCTTTTTCCTAGTA(t/a)CCTTTGCTCTGGGATTGTTAAGAT

JAX Protocol

Protocol Primers

Primer	5' Label	Sequence 5' → 3'	3' Label	Primer Type	Reaction	Note
11261		GAC ATC CGT AGG GCC TTT TTC
11262	Fluorophore	GCC TCA ATC ACC ACC ATT ACG
11263		GGG CCT TTT TCC TAG T

Reaction A

Component	Final Concentration
ddH2O
Kapa 2G HS buffer	1.00
MgCl2	2.00
dNTPS-kapa	0.20
11261	0.50
11262	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.