Genotyping Protocols Database
Protocol 33009: Probe Assay - App<em2Adiuj> (91bp del)
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

Mut= 99 bp

Wt= 100 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletions in lower case:

 AAGGCAGTTTTTGCCTCCTTGTGGCTGGCGGTCACACTAACGGATGGCCCTGCATACTTTGTGtttgacgcaggttctgggctgacaaacatcaagacggaagagatctcggaagtgaagatggatgcagaattcggacatgattcaggatttgAAGTCCGCCATCAAAAACTGGTAGGCAAAAATAAAC

91bp del

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
40445 AAG GCA GTT TTT GCC TCC TT Common A
40446 CCG TCT TGA TGT TTG TCA GC Wild type Reverse A
40447 Fluorophore-1 CTT TGT GTT TGA CGC AGG TTC Quencher-1 WT Probe
40448 Fluorophore-2 CCC TGC ATA CTT TGT GAA GTC C Quencher-2 MUT Probe
40449 GTT TAT TTT TGC CTA CCA GTT TTT G Mutant Reverse A

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
40445 0.40 uM
40446 0.40 uM
40449 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.