Protocol 32948: QPCR Assay - Tg(Ckmm-cre)5Khn qPCR
Version 1.0

Notes

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=  126 bp

IPC = 74 bp

Sequence

Tg Sequence: agcaactgagttcttaagtctgaacccgGTCTGCTCacagggtcccaaaggccgccggcctcgaggtcgacggtatcgataagcttgatatcgacgccctcgaccatgcccaagaagaagaggaaggTGTCCAATTTACTGACCGTACACCAAAATTTGCCTGCATTACCGGTCGATGCAACGAGTGATGAGGTTCGCAA

 

 

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
38995 AGT CTG AAC CCG GTC TGC T Transgene Forward A
38996 TCA GTA AAT TGG ACA CCT TCC TC Transgene Reverse A
38997 Fluorophore-1 CCT CGA GGT CGA CGG TAT CGA TAA GC Quencher-1 Tg Probe
oIMR1544 CAC GTG GGC TCC AGC ATT Internal Positive Control Forward A
oIMR3580 TCA CCA GTC ATT TCT GCC TTT G Internal Positive Control Reverse A
TmoIMR0105 Fluorophore-2 CCA ATG GTC GGG CAC TGC TCA A Quencher-2 IC Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
38995 0.40 uM
38996 0.40 uM
oIMR1544 0.40 uM
oIMR3580 0.40 uM
Tg Probe 0.15 uM
IC Probe 0.15 uM
DNA

Cycling

Step Temp °C Time Note
1 95.0 --
2 95.0 --
3 60.0 -- repeat steps 2-3 for 40 cycles
JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.

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
3 strains use this protocol