Stock No: 001026
Protocol 26975: Sanger sequencing Assay - H2<d> rs50413633
Version 1.2

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

Mut=T/T (H2<d>)

WT= A/A (other haplotypes)

Sequence

Aaatactgatcatgcaatggctgggc(A/T)gaagagagaata

gggtgGGACATCTGCCAATCA

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
20229 ACA TCT CTC TGC CCT TTC CA Forward A
20230 TGA TAT GGC TCA TTT GGC TTC Reverse A

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTPS-kapa 0.26 mM
20229 0.50 uM
20230 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
000461 B10.D2-Hc0 H2d H2-T18c/oSnJ
000463 B10.D2-Hc1 H2d H2-T18c/nSnJ
001026 BALB/cByJ
002845 C.B-H2b Tg(H2-Dd)D8Gja/LilJ
000666 C57BL/10SnJ
5 strains use this protocol