Protocol 24076: Standard PCR Assay - Generic mKate2
Version 1.2

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 = 176 bp

Internal positive control = 324 bp

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
16492 CCA CCT GAT CTG CAA CTT GA Transgene Forward A
16493 TGC TAG GGA GGT CGC AGT AT Transgene Reverse A
oIMR7338 CTA GGC CAC AGA ATT GAA AGA TCT Wild type Forward A
oIMR7339 GTA GGT GGA AAT TCT AGC ATC ATC C Wild type Reverse A

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
16492 0.50 uM
16493 0.50 uM
oIMR7338 0.50 uM
oIMR7339 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
033544 B6;129S-Gt(ROSA)26Sortm1(CAG-cre*,-mKate2)Yzwa/J
034111 B6;129S6-Igs7tm193(CAG-EGFP,CAG-tdTomato)Hze/J
021226 STOCK Tg(Thy1-Brainbow3.1)18Jrs/J
021225 STOCK Tg(Thy1-Brainbow3.1)3Jrs/J
021227 STOCK Tg(Thy1-Brainbow3.2)7Jrs/J
5 strains use this protocol