Stock No: 000465
Protocol 27704: Sanger sequencing Assay - H2-T18<a/b>-Alternate 3
Version 1.2

Notes

H2-T18<a> = C (Jrs 469, 4804, 465) = "hom"

H2-T18<b>= T (Jrs 666, 664, 457, 438) = "wt"

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

Tctgatgagttattgatctagctatgataa(T/C)cagtgattg(ATTG/----)ctattat(A/C)acagtcaca

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
25450 TGT TTC AGA TTT CTT CCC TCT C Forward A
25451 TAG AAA AGG CAG CAG ACT CG Reverse A

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTPS-kapa 0.26 mM
25450 0.50 uM
25451 0.50 uM
Glycerol 6.50 %
Dye 1.00 X
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
000469 B10.A-H2a H2-T18a/SgSnJ
000465 B10.BR-H2k2 H2-T18a/SgSnJ
004804 B10.BR-H2k2 H2-T18a/SgSnJJrep
000457 B10.RIII-H2r H2-T18b/(71NS)SnJ
4 strains use this protocol