Protocol 31469: Sanger sequencing Assay - Rax<ey1>

ZRDCT Rax^ey1/ChUmdJ

Stock No: 005004

Version 1.0

Notes

Mut = T

WT= A

>chr18:66098359-66098710 352bp CTAAACTTGCAGCTCCAGCA GCTTCGATGCTGTGCAAAC

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

TGAGCCGTTGCCCAAGCCGGTTGCCCGCCGAGTTTAGCTTTGGACAGACCGATCCACCGAGTTTAGCTTTGGACCGACCCATCCATTTGCTCACGTTCTAGAAGTGCACACTTGAGGGGGTGGAGGAGGAGAAGGGAACCGGGCATCGAGCTCCAGTTTGCAAAGTGCACTCCCTCCTCTCCATGCACCTGCCGGGCTGCGCGCCAGCC(A/T)TGGCGGACGGGAGCTTCTCGCTCGCTGGCCACCTGCTCCGCAGCCCGGGCGGGAGCACGTCGCGTTTGCACAGCATCGAAGCCATCCTGGGCTTTACCAAGGAAGACGGCATCCTAGACACCTTTCCTGCAGAGAGGAGCTCCAGGAGCTCGAAAGAGCGGGATCCCAGGCTGGGCGCG

JAX Protocol

Protocol Primers

Primer	5' Label	Sequence 5' → 3'	3' Label	Primer Type	Reaction	Note
36939		CTA AAC TTG CAG CTC CAG CA		Forward	A
36940		GCT TCG ATG CTG TGC AAA C		Reverse	A

Reaction A

Component	Final Concentration
ddH2O
Kapa 2G HS buffer	1.30 X
MgCl2	2.60 mM
dNTPS-kapa	0.26 mM
36939	0.50 uM
36940	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
005005	ZRDCT Rax^+/ChUmdJ
005004	ZRDCT Rax^ey1/ChUmdJ
2 strains use this protocol