Protocol 29084: Standard PCR Assay - Tg(Ckmm-cre)5Khn
Version 3.3


Melting curve analysis is done using a Roche Light Cycler 480.

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 Tm = 85.3°C
Internal positive control Tm = 79.3°C

TG = 455bp

IPC = 324bp

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
oIMR1085 GTG AAA CAG CAT TGC TGT CAC TT Transgene Reverse A
oIMR6754 TAA GTC TGA ACC CGG TCT GC Transgene Forward A
oIMR7338 CTA GGC CAC AGA ATT GAA AGA TCT Internal Positive Control Forward A
oIMR7339 GTA GGT GGA AAT TCT AGC ATC ATC C Internal Positive Control Reverse A

Reaction A

Component Final Concentration
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
oIMR1085 0.50 uM
oIMR6754 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


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
029100 B6.Cg-Fxnem2.1Lutzy Tg(Ckmm-cre)5Khn/J
029720 B6.Cg-Fxnem2Lutzy Fxnem2.1Lutzy Tg(Ckmm-cre)5Khn/J
006475 B6.FVB(129S4)-Tg(Ckmm-cre)5Khn/J
006405 FVB-Tg(Ckmm-cre)5Khn/J
4 strains use this protocol