Protocol 36391: Probe Assay - Rab33b<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

>chr3:51493701+51493808 108bp CTAAAGAGCCACAAGCCATTG CACACCTATGACTCAGCACCA

Mut = 85 bp

Wt = 108 bp

Fam = Mut

Hex = Wt

Sequence

Wt Sequence (deletions in lower case):

 GGCCCAGAGGTCAGAGGTACTTGATTTCTGCATCCCACAGGTCAGTGGACAACCAACTCCTGAGCTGATCTCTGACCTGCATGCATTCAGCATGGCGTGTGTGCACCTGTGTGCACACGGGATAAAAAAACTAAAACTGCTTTTAATCCCAGTACATTTGACTCAGCATGGGCCTCTCATAGCACTAGGCCATAGGTATTCTAAATGGTTTTTGTGTGTTGTATCCTTTTCCCGCAGTGATTTTTAAAAACATTTTAAAAGCCCATACCTGTTCAGATTTATAATCTGTATTTAGTAGTCACATTGTGACTAGACTAGGATTATGAAGCGTGGACCCTGCAGGTAAAGACGGGCCAGAGAGCGGAGAGGTGTTACGGGCAGGAACAGTGGCTGAGTTGCTCTGTTTGTGCCATTGGACTGAGCTTGTCGACTTTGCTTTCTGTTTTCCTCTCCagatccagttgtgggacacggcagggcaggagcggttcaggaagagcatggtccagcactactacaggaatgtgcatgctgtcgtctttgtgtatgacatgaccaacatggccagcttccacagcctgccggcttggatagaggaatgcaaacagcacttgctggcgaacgacatacctcgaattctggtgggaaataaatgtgacttgagaagcgccattcaggtgcccacagacttggcacagaagtttgctgacacacacagtatgcctttgtttgagacctctgctaaaaaccccaatgataatgaccacgtagaagctatattcatgaccttagctcataagctaaagagccacaagccattgatgcttagccagctgcctgataacagaattagcctgaaacctgagaccaagcctgcggtgACTTGCTGGTGCTGAGTCATAGGTGTGGCCATTGCGGGTCTGACTTACCTGAAGAGATGATTGGGAAGTGTGACAGTGATGAAGTTCGTAAGAGTAAATCCTAACTGTAGTAGATCATTCTGCTGTTATCGTTGTTATGATGCCTTTTGTATTTTGCATCCTCTCAATCAAGTTTGTCACTGAGACAGCACATGGAAAAGGCCGCTTTACAGTGGGGATTGAAAATGAAGACTACCGGGGCCATCTTTCCAGCTCCAGCCAGAAGCAGCAGCTTCAAGTGAGCATGATGGACCTTAAATGGTTGGTCCTCTTCCTCTCTGGGTCCCTGGAGCAGCAGAAGCCATGCTGGGTAGTGC

This mutation is a 428 bp deletion beginning at Chromosome 3 position 51,493,354 bp and ending after 51,493,781 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
48622 CAC ACC TAT GAC TCA GCA CCA Common A
48623 CTA AAG AGC CAC AAG CCA TTG Wild type Forward A
48624 TTG CTC TGT TTG TGC CAT TG Mutant Forward A
48625 Fluorophore-1 CAG AAT TAG CCT GAA ACC TGA GAC Quencher-1 WT Probe
48626 Fluorophore-2 TGC TTT CTG TTT TCC TCT CCA Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
48622 0.40 uM
48623 0.40 uM
48624 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.