Genotyping Protocols Database
Stock No: 012706
Protocol 20513: Standard PCR Assay - Cck<tm1.1(cre)Zjh>
Version 4.0

Notes

This assay does not work well without the use of a Hotstart Taq polymerase.

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 = ~215 bp
Heterozygote = ~215 bp and 468 bp
Wild type = 468 bp

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
11214 GAG GGG TCG TAT GTG TGG TT Common A
11215 GGG AGG CAG ATA GGA TCA CA Wild type Forward A
9989 TGG TTT GTC CAA ACT CAT CAA Mutant Forward A

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
11214 0.50 uM
11215 0.50 uM
9989 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
012706 STOCK Ccktm1.1(cre)Zjh/J
019021 B6N.Cg-Ccktm1.1(cre)Zjh/J
2 strains use this protocol