Stock No: 026703
Protocol 27476: Standard PCR Assay - Generic IRES
Version 1.2

Notes

This assay will NOT distinguish hemizygous from homozygous transgenic animals.

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

Transgene = 187 bp

Internal positive control = 415 bp

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
21238 CTG TCC CTG TAT GCC TCT GG Internal Positive Control Forward A
21239 AGA TGG AGA AAG GAC TAG GCT ACA Internal Positive Control Reverse A
24712 CTT GGA ATA AGG CCG GTG T Transgene Forward A
24713 AGC TTC CAG AGG AAC TGC TTC Transgene Reverse A

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
21238 0.50 uM
21239 0.50 uM
24712 0.50 uM
24713 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
031824 B6(FVB)-Tlr4tm1.1Gbrt/J
033394 B6.129-Ntsr2tm1.1(cre)Giml/J
031559 B6.Cg-Crhtm1.1(flpo)Bsab/J
030211 B6.Cg-Npytm1.1(flpo)Hze/J
030849 B6.Cg-Traf6tm2.1Ywc/J
016237 B6.Cg-Tg(Col1a2-cre/ERT,-ALPP)7Cpd/J
017580 C.129S4(B6)-Ifngtm3.1Lky/J
031289 STOCK Lmx1btm2(cre/ERT2)Rjo/J
029803 STOCK Mki67tm2.1(cre/ERT2)Cle/J
029429 STOCK Olfr151tm26Mom Tg(Olfr151,-MAPT/mCherry)5MGaFeins/J
006677 STOCK Olfr151tm28(cre)Mom/MomJ
026703 STOCK Vsx1tm2Mci/ChowJ
026707 STOCK Tg(Grm6-lacZ)1Chow/J
018281 STOCK Tg(Wnt7a-EGFP/cre)#Bhr/Mmjax
14 strains use this protocol