Protocol 33046: Probe Assay - D5Ertd579e<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

>chr5:36619599-36619723 125bp GGATCGAGACTTTAGTAGAAGAGC GGCTTGGTGACACACTCTACA

Mut= 120 bp

Wt= 125 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletions in lower case; and insertions with carrots ^g^ (CCAAG insertion):

CAGGACAGCCAGGGCTACACAGAGAAATCATGTATTGAAAAACCAAAAAAGAAAAAAGAAAAGAAAAGAAAAAATACTTACAGTAGGTTAGCTTTACCGgtttttaaactctgggattcatttaagaattgataatatattccttttggaacatggaaacttgtggtcctccgtagtgacataaaataattgcctgtctactgtgcctggtagaaagctgactgtcttctctctcacacagagctctgggatcgagactttagtagaagagctctgctgcagactgaaagacctacagagcgagcaaggtgagcaggctggggcaggtagagccctacaggGtccttctcagtgtagagtgtgtcaccaagccAGCATTCCTCATTACCTCAGCTCTGCACAAGGCTTCAGTGAAGTCCTTAGTGGCCACATGGGAAATCCCTGCTCCAGAGGAGGGCTGTAGTCCGTGGGCACCTTCTAAGGCTTCACTTGGGGGTTCAGTCATTTAAGTTCCTAAGTGTTCTGCAGGACTCCAGGAAGGGGCTGCATACTCTGAAACCC^ttttctgtccttgtcaa^GGTCATTGTCAGCGTATACTCTTGAAGCCTTGTGTTAAGGGTCCACCTTATTTT

This mutation is a 242 bp deletion beginning at Chromosome 5 position 36,619,631 bp and ending after 36,619,872 bp (GRCm38/mm10).  In addition, there is a (CCAAG) 5 bp insertion and a 17 bp deletion (TTTTCTGTCCTTGTCAA) 220 bp after the 242 bp deletion that will not alter the results of the exon deletion.

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
40530 GGA TCG AGA CTT TAG TAG AAG AGC Wild type Forward A
40531 GGC TTG GTG ACA CAC TCT ACA Common A
40532 GGG CTA CAC AGA GAA ATC ATG T Mutant Forward A
40533 Fluorophore-1 CTG CTG CAG ACT GAA AGA CCT A Quencher-1 WT Probe
40534 Fluorophore-2 TTA GCT TTA CCG GTC CTT CTC A Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
40530 0.40 uM
40531 0.40 uM
40532 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.