Protocol 13575: Pyrosequencing Assay - Tyr<c-2J>
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

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
9581 GCA CCA TCT GGA CCT CAG TTC Forward
9582 Fluorophore ATA AAA CAC AGA GGG CCA GGA CT Reverse
9583 AAA GGG GTG GAT GAC SEQ

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.00
MgCl2 2.00
dNTPS-kapa 0.20
9581 0.50
9582 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
000058 B6(Cg)-Tyrc-2J/J
033605 B6.Cg-S1pr1tm4.1Rlp Gt(ROSA)26Sortm2.1(luc*/Arrb2*)Rlp Tyrc-2J/J
023428 B6;129X1-Tyrc-2J Cdkn1atm2Hpw/J
027662 C57BL/6-Tyrc-Brd Tg(Gnrhr-luc/EGFP)D8Mrln/J
026622 NOD.Cg-KitW-41J Tyr + Prkdcscid Il2rgtm1Wjl/ThomJ
5 strains use this protocol