Protocol 49107: Probe Assay - Tg(Plp1-cre/ERT)3Pop-Probe
Version 2.0

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

Tg=  127 bp

IPC = 74 bp

Sequence

Tg Sequence:

CTTGGTAACATGGGCTGCTTGGCCCAGCAGTATAGTGTGAGTGGATGAGTTACCTCGTATGCGTACCTGACTTTCTCCTTCTTCTTCCCCAGGCTggccagagtggcccgggggcgcgccggatctcgacatcgtaagcttctgcagcccaattccaccATGTCCAATTTACTGACCGTACACCAAAATTTGCCTGCATTACCGGTCGATGCAACGAGTGATGAGGTTCGCAAGAACCTGATGGACATGTTCAGGGATCGCCAGGCGTTTTCTGAGCATACCTGGAAAATGCTTCTGTCCGTTTGCCGGTCGTGGGCGGCATGGTGCAAGTTGAATAACCG

 

 

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
74489 CGT ATG CGT ACC TGA CTT Transgene Forward A Plp1
74490 TGT ACG GTC AGT AAA TTG GA Transgene Reverse A Cre-ERT
74491 Fluorophore-1 TTC TGC AGC CCA ATT CCA CC 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
74489 0.40 uM
74490 0.40 uM
oIMR1544 0.40 uM
oIMR3580 0.40 uM
Wt Probe 0.15 uM
Mutant Probe 0.15 uM
DNA

Cycling

Step Temp °C Time Note
1 95.0 --
2 95.0 --
3 60.0 --
4 -- repeat steps 2-3 for 40 cycles
5 40.0 -- Forever
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
005975 B6.Cg-Tg(Plp1-cre/ERT)3Pop/J
024701 D2.Cg-Tg(Plp1-cre/ERT)3Pop/SjJ
2 strains use this protocol