Stock No: 011122
Protocol 21773: Probe Assay - Mertk<tm1Grl> Probe
Version 1.0

Notes

Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination. The transgene genotype is determined by comparing ΔCt values of each unknown sample against known homozygous and hemizygous controls, using appropriate endogenous references.

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

Mutant=  103 bp

Wild Type = 119 bp

Sequence

Wt Sequence: tgccattatacctggctttcattttttttccattaattaatttatttattcactttacatagcatctttaattcaggacaACTAGTacgatgtggtagcgtactcacatttgctttgca

 

Mutant Sequence: TGCCAAGTTCTAATTCCATCAGAAGCTGACTCTAGGAAAAGAGGACTGCGTGTGGGAAGAGAAGTctagtacgatgtggtagcgtactcacatttgctttgca

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
18138 TGC CAT TAT ACC TGG CTT TCA Wild type Forward A
27228 TGC CAA GTT CTA ATT CCA TCA G Mutant Forward A
33212 TGC AAA GCA AAT GTG AGT ACG Common A
33213 Fluorophore-1 CAT AGC ATC TTT AAT TCA GGA CAA CTA G Quencher-1 WT Probe
33214 Fluorophore-2 CTG CGT GTG GGA AGA GAA G Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
18138 0.40 uM
27228 0.40 uM
33212 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 4.0 -- Forever
JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.

Strains Using This Protocol

This is the only strain that uses this protocol.