Stock No: 000461
Protocol 14434: Pyrosequencing Assay - Hc<0>
Version 2.0

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

WT = TA/TA

HET= --/TA

Mut = --/--

Sequence

ACAACAACTGGAACTGCA[ta]CTTTGAAATTAAAGAATA

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
11590 ATT TTG CTT CCA CAG GTA TGG T
11591 Fluorophore TGA CAG GGC AAG TTA GAA CTA AGC
11592 ACA ACA ACT GGA ACT GC

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.00
MgCl2 2.00
dNTPS-kapa 0.20
11590 0.50
11591 0.50
Glycerol 5.00
Kapa 2G HS taq polym 0.01
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
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.

Strains Using This Protocol

Stock Number Strain Name
000461 B10.D2-Hc0 H2d H2-T18c/oSnJ
000463 B10.D2-Hc1 H2d H2-T18c/nSnJ
2 strains use this protocol