Genotyping Protocols Database
Stock No: 010908
Protocol 29387: Standard PCR Assay - Vip<tm1(cre)Zjh>
Version 3.2

Notes

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

Changed expected mutant band size from ~250 to ~350, JAK 2/18/13.

 

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 ~350 bp
Heterozygote = ~350 bp and 175 bp
Wildtype = 175bp

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
9527 CCC CCT GAA CCT GAA ACA TA Mutant Forward A
9984 GGA CAC AGT AAG GGC ACA CA Common A
9985 TCC TTG GAA CAT TCC TCA GC Wild type Forward A

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
9527 0.50 uM
9984 0.50 uM
9985 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
010908 STOCK Viptm1(cre)Zjh/J
031628 B6J.Cg-Viptm1(cre)Zjh/AreckJ
2 strains use this protocol