Protocol 33396: Probe Assay - Stt3b<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

>chr9:115280591-115280698 108bp CCTTAAACCTTGAAAACTGGTTC TGGCTACACATATCTCACTATGAAG

Mut= 121 bp

Wt= 108 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

TCTTGATTTCTTAACTTTTTCATACGTTGTTACCTTGTAATTTGCCCTTCACTTTTGTCTGTTAAAATAACCTGCTTATGATTTAACTGGATAATCACAGTTTTCCTTAAACCTTGAAAACTGGTTCTTTTATCATCTGTTCATCACAGTAgtagcctcttcacagggacttgcttcacagttctctcttcatagtgagatatgtgtagccatatattgaatataaagaaaggtgtccttgtgttaactctttttataacatttcctcttctcttttgtccaggtttaactatagatcaacacatcatcttgcatctcatggattctatgagtttctaaattggtttgatgaaagagcatggtacccactgggaagaatagtgggtggcaccgtaagtatattccttacctttgcggtaactgtaaactgtggagtaaagcatttcctgctaagggacgagggtctcggaaggccgtcattagttcacagacacatcttccatggctaattagccagtgtttctcagctccgctcttggcagtttatttgtaggatatcttacactgccccccatttctgattgaggtcaggggctggattaagaatctgctgtcacaaagtacctcaagtccagagtgcttttcgctttgtttctgaacaattgagtttaaaagcttttgggggaaggaaataaaaattttgaaaatcaaattaattaaatgtttccagcctcaagcctctgaattaatgtagataattgaaccgtattgagtagggtagagtGTTCTGCCTCCAGAAGGATGACATGAAGGTTCTTAGGGTTCAGTGTGTAAGGAGAACTCCTAGAGAAGGGGGAAGATGTGGCAGGCAGCTCTCGGAGAAGTTTGTACACTTTCGTTTCTTCTTGAATTT

This mutation is a 633 bp deletion beginning at Chromosome 9 position 115,280,019 bp and ending after 115,280,651 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
41319 CCT TAA ACC TTG AAA ACT GGT TC Common A
41320 TGG CTA CAC ATA TCT CAC TAT GAA G Wild type Reverse A
41321 TTC CCC CTT CTC TAG GAG TTC Mutant Reverse A
41322 Fluorophore-1 CAC AGG GAC TTG CTT CAC AGT Quencher-1 WT Probe
41323 Fluorophore-2 CAT CAC AGT AGT TCT GCC TCC A Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
41319 0.40 uM
41320 0.40 uM
41321 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.