Stock No: 005698
Protocol 20363: Standard PCR Assay - Tg(Gfap-TK)7.1Mvs Alternate2
Version 1.0

Notes

This assay will NOT distinguish hemizygous from homozygous transgenic animals.

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

Transgene = 290 bp

Internal positive control = 190 bp

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
25783 GTC AAC GGG GGA CAT AAA AG Internal Positive Control Forward A
25784 CTT CTA AGA GTT AAA CAA TAC CAG CTT Internal Positive Control Reverse A
30290 GGG GCC TCG GTC CTA GT Transgene Forward A
30291 GAT GGC AGG GGT ACG AAG 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
25783 0.50 uM
25784 0.50 uM
30290 0.50 uM
30291 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
017523 129S6.Cg-Tg(Gfap-TK)7.1Mvs/RhnJ
005698 B6.Cg-Tg(Gfap-TK)7.1Mvs/J
2 strains use this protocol