Protocol 27167: Standard PCR Assay - Generic Cre/ERT2 alternate1
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 = ~200 bp

Internal positive control = 324 bp

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
19974 GGC CAG GCT GTT CTT CTT AG Transgene Reverse A ERT2
oIMR7338 CTA GGC CAC AGA ATT GAA AGA TCT Internal Positive Control Forward A
oIMR7339 GTA GGT GGA AAT TCT AGC ATC ATC C Internal Positive Control Reverse A
oIMR8346 ATA CCG GAG ATC ATG CAA GC Transgene Forward A Cre

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
19974 0.50 uM
oIMR7338 0.50 uM
oIMR7339 0.50 uM
oIMR8346 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
026802 B6(Cg)-Ins1tm2.1(cre/ERT2)Thor/J
016237 B6.Cg-Tg(Col1a2-cre/ERT,-ALPP)7Cpd/J
005975 B6.Cg-Tg(Plp1-cre/ERT)3Pop/J
029211 B6.Cg-Tg(Prrx1-cre/ERT2,-EGFP)1Smkm/J
020282 B6.Cg-Tg(Vil1-cre/ERT2)23Syr/J
029231 B6;129-Bhlha15tm4Skz/J
030346 B6;129S4-Gcgem1(cre/ERT2)Khk/Mmjax
014160 B6;DBA-Tg(S100b-EGFP/cre/ERT2)22Amc/J
033019 B6N.Cg-Tg(Vil1-cre)20Syr/J
024760 FVB/N-Tg(Ddx4-cre/ERT2)1Dcas/J
024283 STOCK Hcn4tm2.1(cre/ERT2)Sev/J
025736 STOCK Tg(Cela1-cre/ERT)1Lgdn/J
028364 STOCK Tg(Neurod1-cre)1Able/J
13 strains use this protocol