Genotyping Protocols Database
Stock No: 002448
Protocol 26670: Sanger sequencing Assay - H2<b>rs253049200
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

Sequence

AAGCAGACACAGGAGAGAGGATGTTCTGTaaaacaagcatgtga

Aagga(tg/--)cg(atg/---)cgtgatgaaggattctttgctaaaGACACA

ATTATGGATCTGCAA

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
20197 AGT CGG TTG GAT GGT GTT GT Forward A
20198 AAT GGG CCT CTC TTT CCA GT Reverse A

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTPS-kapa 0.26 mM
20197 0.50 uM
20198 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
000438 C3.SW-H2b/SnJ
000664 C57BL/6J
000666 C57BL/10SnJ
002448 129S1/SvImJ
002591 NOD.B10Sn-H2b/J
002845 C.B-H2b Tg(H2-Dd)D8Gja/LilJ
005304 C57BL/6NJ
7 strains use this protocol