Protocol 36154: Probe Assay - Bcap29<em1(IMPC)J>Alternate2
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

>chr12:31626957-31627068 112bp CCACTACTATCCATTTACAAACGT CCAGCCTTTTAAATAATGGACTTTAG

 Mut= 113 bp

Wt= 112 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletions in lower case):

 AGGCTGGATTATATCTGTTGCTGACAGCAAGTTGGATTTTAGTCATGCATAGAAATGGAAAGAGTCAGAGATAATATTTCACAGTAGATCAAGACTTGACAGTAAGATGATAAGTGAGCCCCAGACGAATGATTTTAAAATGCTATAGTGCTGATTAATAACTTGCCACTACTATCCATTTACAAACGTCCATCTTTAAGCTCAATACCCTGTCAGGTACtaaagagggacagacagaaagaaaggcatatctaaagtccattatttaaaaggctggtaggttattctacaactccttcttgtttaaagaaaatgctttcttaaaaactaaaagtagcgtcctctgtcttgtaacagatgccgtgagagaagtaagaaagtattcgtccacgaatgttgtagagaagaactcagccatcagaccgagtgcctttgagcacacacagatgaagctcttcaggtctcaaagaaatctgtatatttctggattctcattatttttttggCTGtaagtaaaaaaaaatttgctagcagttgtatcactcatagaactcccctttgttttttccacttacACTAAGATCTTTTCATTGATATCTTCATAAAAGAATGCAAATGATACACTCAAGTAACAAATCTCAGTCACACTAAAGAACGCTTGGAATGGAAGATGTTGCCTTTGGAAGGGAAGAGCCCATGAATTGGTTGTCCAATGCCAAATGTTCAGCCCTGAAAACATACATCCAGGTAGCAGTATATGGACTGAACAGGCTATATTTAGGAATATATATGTATG

This mutation is a 355 bp deletion beginning at Chromosome 12 position 31,626,659 bp and ending after 31,627,013 bp (GRCm38/mm10).In addition, there is a 3 bp endogenous retention (CTG) 286 bp after the start of the deletion and then an additional 66 bp after the 3 bp retention.

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
46975 CCA GCC TTT TAA ATA ATG GAC TTT AG Wild type Reverse A
46976 GTT ACT TGA GTG TAT CAT TTG CAT TC Mutant Reverse A
46977 Fluorophore-1 AGG GAC AGA CAG AAA GAA AGG Quencher-1 WT Probe
47858 CCA CTA CTA TCC ATT TAC AAA CGT Common A
47917 Fluorophore-2 AGG TAC CTG ACT AAG ATC TTT TCA TTG Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
46975 0.40 uM
46976 0.40 uM
47858 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.