Protocol 35820: Probe Assay - Grn<tm1.1Far> 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

Mutant=  109 bp

Wild Type = 122 bp

>chr11:102432793+102432914 122bp CACACTGGTAGGACGCTGA TCTACTCTGAGGCCACGCACT

Sequence

Wt Sequence: gccagctgacgcatgtgcgcttgctgcacactggtaggacgctgacacaagcgacacatctgcttgctttcctgttggcttgttgagaagactcaatgaagccagggccttggtcagagcacaggggagtgcgtggcctcagagtagaaggtggcacagagtttaaggtggctgccaaactttgcttggcatttgtagGCAGACCATGTGGGTCCTGA

 

Mutant Sequence: TGACGCATGTGCGCTTGCTGCACACTGGTAGGACGCTGACgtcgacATAACTTCGTATAGCATACATTATACGAAGTTatattaagggttccggatcaacgaagttcctattccgaagttcctattctctagaaagtata

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
40717 GAG AAT AGG AAC TTC GGA ATA GG Mutant Reverse A
47089 CAC ACT GGT AGG ACG CTG A Common A
47090 Fluorophore-1 AAG GGT TCC GGA TCA ACG AAG T Quencher-1 MUT Probe
47091 TCT ACT CTG AGG CCA CGC ACT Wild type Reverse A
47092 Fluorophore-2 AGA CTC AAT GAA GCC AGG GCC T Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
40717 0.40 uM
47089 0.40 uM
47091 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.