Protocol 34242: Probe Assay - Cmah<em1Guru> 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 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

>chr13:24422533+24422630 98bp GCCATCACACTTCATCAGCA CTTCCAGTTGTGCTTTGTGC

Mutant=  115 bp

Wild Type = 98 bp

Sequence

Wt Sequence: gccatcacacttcatcagcactaatttttttcactgtgtagtacaactgactctcatttccccccagGTCCGTTAAATGCACAAAGCACAACTggaag

 

Mutant Sequence: gccatcacacttcatcagcactaatttttttcactgtgtagtacaactgactctcatttccccccaGGAAGTTAGACGTGAGCACCATGAAATATATCAACCCTCCAGGGAGCTT

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
14414 CTT CCA GTT GTG CTT TGT GC Wild type Reverse A
43144 GCC ATC ACA CTT CAT CAG CA Common A
43145 AAG CTC CCT GGA GGG TTG A Mutant Reverse A
43146 Fluorophore-1 TTT CCC CCC AGG TCC GT Quencher-1 WT Probe
43147 Fluorophore-2 CCC CCA GGA AGT TAG ACG T Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
14414 0.40 uM
43144 0.40 uM
43145 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

This is the only strain that uses this protocol.