Protocol 13575: Pyrosequencing Assay - Tyr<c-2J>

C57BL/6-Tyr^c-Brd Tg(Gh1-luc/EGFP)D8Mrln/J

Stock No: 027662

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

JAX Protocol

Protocol Primers

Primer	5' Label	Sequence 5' → 3'	3' Label	Primer Type	Reaction	Note
9581		GCA CCA TCT GGA CCT CAG TTC		Forward
9582	Fluorophore	ATA AAA CAC AGA GGG CCA GGA CT		Reverse
9583		AAA GGG GTG GAT GAC		SEQ

Reaction A

Component	Final Concentration
ddH2O
Kapa 2G HS buffer	1.00
MgCl2	2.00
dNTPS-kapa	0.20
9581	0.50
9582	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
036977	B6(Cg)-Dbp^em1Drw Tyr^c-2J/J
000058	B6(Cg)-Tyr^c-2J/J
036153	B6.Cg-Gt(ROSA)26Sor^em7Mvw Tyr^c-2J/MvwJ
035186	B6.Cg-Htra3^tm1Sud Tyr^c-2J/J
029233	B6.Cg-Kit^tm1.1Sraf/Mmjax
033605	B6.Cg-S1pr1^tm4.1Rlp Gt(ROSA)26Sor^tm2.1(luc*/Arrb2*)Rlp Tyr^c-2J/J
023428	B6;129X1-Tyr^c-2J Cdkn1a^tm2Hpw/J
027662	C57BL/6-Tyr^c-Brd Tg(Gh1-luc/EGFP)D8Mrln/J
026622	NOD.Cg-Kit^W-41J Tyr ^+ Prkdc^scid Il2rg^tm1Wjl/ThomJ
9 strains use this protocol