Stock No: 003528
Protocol 27450: Standard PCR Assay - Tg(GFAP-TVA)5Hev Alternate1
Version 1.2

Notes

This assay cannot distinguish hemi from hom

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 = 191 bp
IPC = 217 bp

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
21221 GAC AAA ATG GTG AAG GTC GG Internal Positive Control Forward A
21222 CAA AGG CGG AGT TAC CAG AG Internal Positive Control Reverse A
23942 CAG ATT TGA GAG CTC ATG TCC A Transgene Forward A
23943 ACG GAC AAC GGC ACA GAG Transgene Reverse A

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
21221 0.50 uM
21222 0.50 uM
23942 0.50 uM
23943 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
003528 STOCK Tg(GFAP-TVA)5Hev/J
003529 STOCK Tg(NES-TVA)J12Ech/J
2 strains use this protocol