Protocol 5990: Standard PCR Assay - Tg(Sry)Eicher
Version 1.0

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

Mutation =  350 bp
Transgene = 420 bp
Internal Positive Control = 200 bp

XX female= 200 bp
XY- female= 350 bp and 200 bp
XX male= 420 bp and 200 bp
XY- male = 420 bp and 350 bp and 200 bp

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
9369 CTG GAG CTC TAC AGT GAT GA Forward
9370 CAG TTA CCA ATC AAC ACA TCA C Reverse
9371 AGC CCT ACA GCC ACA TGA TA Transgene Forward Sry
9372 GTC TTG CCT GTA TGT GAT GG Transgene Reverse Sry
oIMR8744 CAA ATG TTG CTT GTC TGG TG Internal Positive Control
oIMR8745 GTC AGT CGA GTG CAC AGT TT Internal Positive Control

Reaction A

Component Final Concentration
ddH2O
KAPA Rxn Buffer Contains 1.5 mM Mg2+ final concentration. 1.00
MgCl2 0.00
dNTP 0.20
9369 1.00
9370 1.00
9371 1.00
9372 1.00
oIMR8744 0.10
oIMR8745 0.10
DNA Loading Dye 0.69
KAPATaq DNA Polymera No proofreading function. 1 error per 40000 nucleotides. Initial denature 2 min. 0.03
DNA

Cycling

Step Temp °C Time Note
1 94.0 --
2 94.0 --
3 60.0 --
4 72.0 --
5 -- Go to step 2-4, 35 times
6 72.0 --
7 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
010905 B6.Cg-Tg(Sry)2Ei Srydl1Rlb/ArnoJ
002063 C57BL/6JEi-Tg(Sry)1Ei Chr YAKR/J/EiJ
002708 C57BL/6JEi-Tg(Sry)2Ei Chr YAKR/J/EiJ
002837 C57BL/6JEi-Tg(Sry)3Ei Chr YAKR/J/EiJ
002183 C57BL/6JEi-Tg(Sry)4Ei Chr YPOS/EiJ
5 strains use this protocol