Stock No: 007924
Protocol 23791: Standard PCR Assay - En2<tm4(cre/ERT2)Alj>
Version 1.2

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 = 397 bp
Heterozygote = 311 bp and 397 bp
Wild type = 311 bp
Separated by gel electrophoresis on a 1.5 - 3% agarose gel.

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
oIMR9115 TGC TCT TTG ACG CTT CGG TG Wild type Forward A
oIMR9116 CCT TGG ATG GAG TGC TCA AAG C Wild type Reverse A
oIMR9117 GCC TGG TCT GGA CAC AGT GCC Mutant Forward A
oIMR9118 CTG TCT GCC AGG TTG GTC AGT AAG C Mutant Reverse A

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
oIMR9115 0.50 uM
oIMR9116 0.50 uM
oIMR9117 0.50 uM
oIMR9118 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

This is the only strain that uses this protocol.