Stock No: 004176
Protocol 14667: Pyrosequencing Assay - Tub<tub>
Version 3.0

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

Mutant = A/A
Heterozygote = A/C
Wild type = C/C

Sequence

WT=G
Mut=T
AACTTCCAGATCATCCACGGCAATGACC(g/t)TGAGTGTTGCCACTCCCTGTTTTTGATGT

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
pIMR62 GGA GTG GCA ACA CTC A SEQ
poIMR0060 Fluorophore ACG CCA GTC TGG AAT GAT GA Forward
poIMR0061 ATA TGG ACC AGA TCA GGG GAT TG Reverse

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.00
MgCl2 2.00
dNTPS-kapa 0.20
poIMR0060 0.50
poIMR0061 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
000562 B6(AU)-Tubtub/J
004176 BKS.B6-Tubtub/Jng
003711 CAST.B6-Tubtub/Jng
3 strains use this protocol