Protocol 26273: Standard PCR Assay - Cyp1a2/Cyp1a1<tm2Dwn>
Version 2.2

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

HET = 353 bp and 417 bp
MUT = 353 bp
WT = 417 bp
Separated by gel electrophoresis on a 1.5% agarose gel.

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
oIMR7515 GGC TAA CCA TCT CGT CAG C A
oIMR7516 TGC ACA CGG CAC TCT GAG A
oIMR7517 GAC ATA GGA GCT ACC TAC AC A
oIMR7518 GTC AAA GTA ACC AGA CAC ATC CTG C A

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
oIMR7515 0.50 uM
oIMR7516 0.50 uM
oIMR7517 0.50 uM
oIMR7518 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
008599 B6.Cg-Cyp1tm1Dwn Ahrd Tg(CYP1A1,CYP1A2)1Dwn/DwnJ
007580 STOCK Cyp1atm1Dwn Tg(CYP1A1,CYP1A2)1Dwn/J
2 strains use this protocol