Stock No: 024842
Protocol 27303: Standard PCR Assay - Tg(Vil1-cre)1000Gum-Alternate 1
Version 2.2

Notes

This assay CANNOT tell the difference between tg/0 and tg/tg. 
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

TG= ~150 bp
Hemizygous = ~150 bp and 182 bp
IPC = 182 bp

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
16775 GCC TTC TCC TCT AGG CTC GT Common A Vil1
16776 TAT AGG GCA GAG CTG GAG GA Internal Positive Control Reverse A Vil1
oIMR9074 AGG CAA ATT TTG GTG TAC GG Transgene Reverse A Cre R

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
16775 0.50 uM
16776 0.50 uM
oIMR9074 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
021504 B6.Cg-Tg(Vil1-cre)1000Gum/J
024842 NOD.Cg-Tg(Vil1-cre)1000Gum/J
2 strains use this protocol