Protocol 36197: Probe Assay - Fam193a<em1(IMPC)J>
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

>chr5:34426472+34426583 112bp CGATCCATCAGTACCTTTCTTG GCTGCATATTATCTCCCTACCA

Mut= 112 bp

Wt= 112 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 ATCTCTGCTGGGATTGGGGCTTGTCACTTTCTAATTCAGTCATTGGATTAGCACCTAGGGTCAATTCTCAGGCACCTTTTACTCTTATTTGTGAGTTATAATACCTTCCTCAGAGATGGACTAAGATCTTTTTTCATGGACTGTAGGTATGGAGTACAGGTTGCATAAACAGTGCAGTCCTAGGAAGCATTTGGGGTCATGTCAGTGTCTATGAGTTACCTGGCATCAAATGCTCTAGGTGCACAAATCCcataggttgtcacaagaaagacagtggattgtcagtgacactgacaggaaaactgcctgttaactgacatgtgtggacaaatcatggctaagtggtgattctttctgtgtctctacaggctctgtgagcgggacccctatcagctctaccagcggttggagcaacaagcccgggaatatgtgctggagatgaaggtccgcctgcttcggcagctctctgctgcagccaaggccaaggcaccgtctggcttgcagggcccacctcaggcacaccactttgtctccctactcctggaggagtatgctgccctttgccaggcagcacgatccatcagtacctttcttggcactctggtgagagaccatatccacttctttcctggcTGCTGAGGTGAACATAAGAACAAAAGCATTTGGTAGGGAGATAATATGCAGCCAGGGGACTGATCATATGGTTAAGCCCTCCTTGTATGATCACAGAGTCCTTTGCACACAAGGTGTGGTAGGATTGTTAGTTTAATGGAATTTGCTAGCAGAGAGAAGCATTTAGAGAATTAATGTCCAGGAGCCATAGGATGAAAATCTGTTTGGGAAAACCTGAGCACTAGGAAGTTAGACACTAGGATATGGAGGTGAGAACAGTGATACA

This mutation is a 383 bp deletion beginning at Chromosome 5 position 34,426,149 bp and ending after 34,426,531 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
48089 CGA TCC ATC AGT ACC TTT CTT G Wild type Forward A
48090 GCT GCA TAT TAT CTC CCT ACC A Common A
48091 TTG GGG TCA TGT CAG TGT CT Mutant Forward A
48092 Fluorophore-1 ACT CTG GTG AGA GAC CAT ATC CAC Quencher-1 WT Probe
48093 Fluorophore-2 CTA GGT GCA CAA ATC CTG CTG Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
48089 0.40 uM
48090 0.40 uM
48091 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.