Protocol 14352: Pyrosequencing Assay - Dmd<mdx-4Cv>

B6.Cg-Terc^tm1Rdp Dmd^mdx-4Cv/BlauJ

Stock No: 023535

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

CAAGAACAGCTGCAGAACAGGAGA(c/t)AACAGTTGAATGAAATGTTAAAG

JAX Protocol

Protocol Primers

Primer	5' Label	Sequence 5' → 3'	3' Label	Primer Type	Reaction	Note
11882		CAG ATT CAG TGG GAT GAG GTT CAA		Forward
11883	Fluorophore	CCT TAG CTT CCA GCC ATT GTG T		Reverse
11884		ACA GCT GCA GAA CAG G		SEQ

Reaction A

Component	Final Concentration
ddH2O
Kapa 2G HS buffer	1.00
MgCl2	2.00
dNTPS-kapa	0.20
11882	0.50
11883	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
023535	B6.Cg-Terc^tm1Rdp Dmd^mdx-4Cv/BlauJ
002378	B6Ros.Cg-Dmd^mdx-4Cv/J
2 strains use this protocol