Protocol 35759: Sanger sequencing Assay - Tyr<c-2J>-SEQ

B6(Cg)-Cd8b1^em1Jmlr Tyr^c-2J/JmlrJ

Stock No: 039964

Version 1.0

Notes

Mut = T

WT= G

>chr7:94641468-94641778 311bp AGAACTTGTTGGCAAAAGAATG CTTTTCGGAGACACTCAAATCA

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

ATGTTCTTGGCTGTTTTGTATTGCCTTCTGTGGAGTTTCCAGATCTCTGATGGCCATTTTCCTCGAGCCTGTGCCTCCTCTAAGAACTTGTTGGCAAAAGAATGCTGCCCACCATGGATGGGTGATGGGAGTCCCTGCGGCCAGCTTTCAGGCAGAGGTTCCTGCCAGGATATCCTTCTGTCCAGTGCACCATCTGGACCTCAGTTCCCCTTCAAAGGGGTGGATGACC[g/t]TGAGTCCTGGCCCTCTGTGTTTTATAATAGGACCTGCCAGTGCTCAGGCAACTTCATGGGTTTCAACTGCGGAAACTGTAAGTTTGGATTTGGGGGCCCAAATTGTACAGAGAAGCGAGTCTTGATTAGAAGAAACATTTTTGATTTGAGTGTCTCCGAAAAGAATAAGTTCTTTTCTTACCTCACTTTAGCAAAACATAC

JAX Protocol

Protocol Primers

Primer	5' Label	Sequence 5' → 3'	3' Label	Primer Type	Reaction	Note
46959		AGA ACT TGT TGG CAA AAG AAT G		Forward	A
46960		CTT TTC GGA GAC ACT CAA ATC A		Reverse	A

Reaction A

Component	Final Concentration
ddH2O
Kapa 2G HS buffer	1.30 X
MgCl2	2.60 mM
dNTPS-kapa	0.26 mM
46959	0.50 uM
46960	0.50 uM
Glycerol	6.50 %
Kapa 2G HS taq polym	0.03 U/ul
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 (Touchdown)
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.

JAX uses a 'touchdown' cycling protocol and therefore has not calculated the optimal annealing temperature for each set of primers.

Strains Using This Protocol

Stock Number	Strain Name
039964	B6(Cg)-Cd8b1^em1Jmlr Tyr^c-2J/JmlrJ
036977	B6(Cg)-Dbp^em1Drw Tyr^c-2J/J
039963	B6(Cg)-Tyr^c-2J H2-Eb1^em1Jmlr/JmlrJ
036153	B6.Cg-Gt(ROSA)26Sor^em7Mvw Tyr^c-2J/MvwJ
039236	B6.Cg-Gt(ROSA)26Sor^tm1.1(CAG-rtTA3,-GFP*,-luc*)Zhrt Tyr^c-2J/ZhrtJ
039259	B6.Cg-Gt(ROSA)26Sor^tm1.2(CAG-rtTA3,-GFP*,-luc*)Zhrt Tyr^c-2J/ZhrtJ
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
017840	B6.Cg-Tyr^c-2J Hr^hr/J
039331	B6;129S6-Rho^tm2.1Kpal 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
039157	STOCK Lcn2^tm2Bara Tyr^c-2J/J
15 strains use this protocol