Protocol 46193: Probe Assay - Bpifb6<em1(IMPC)J> Alt2
Version 3.0

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

Mutant=  112 bp

Wild Type = 146 bp

  
>chr2:153904671+153904816 146bp GTGGTCCTCACTGCTCTTCC TGATGGCTTTGCTGAGACAC

Sequence

Wt Sequence (deletions in lower case):

GGTCCTAGTAGACTGGGCTAGAATCTTACTCAGACCTCTCAGTGAGGTGGTGGGGGTCATGGTGAGGGCCTGGGTCTTGACCTCTCagttctcattgtacatgtgtgtccccctcccagtatgaaggtaaaggacgtcctggagcctgttatcacattgaactttgtacctggagtgggcatctcccagtgtgtgtccacaggcatgaccatcactggcaagaggttggtgtgggagtaaagtggggcggggccgggggagaagaccacacccttgtctcacccttccatacgtaatcactgagtaccggcagttcattatgtgggtgctgggatctgaggtgactgtagacttgggttctgggttgacaggagccaggtgaagcgaggcacagactgaagggggtcggactgagcagtgggggaagaggccagggagagtctggggaggggattacagctgagcagtcttccagatgtgtaagagttcactgacataatgggaatggaaaggttgtttgtgtggctgtgagcaggccccatcctggcttttcaccatgtgtgggtagatcctgccctctgccttctcagattcaaaggttatgggcagatagagtggggatggaagtaccagtagcagagatacccagagtgctgggtcttccagtttcacaggagggaacatggaaatcaacgtggtcctcaacatcacagctactgatcggcttctgcaagatgaggaggcaggcacccctgtgttcaggagtgagggctgtgaggtcatcctggtcagcgtgaaaaccaacctgcctaacaagtaaggtgtcccgcgtctggggagcatggtggtcctcactgctcttccccatcattcctcttcccgagccactgatctcccataaccatgtctctttgtgtcctctgtccaatttggtgccattgatgcactgttcacatggttccccatgcctgtgtctcagcaaagccatcaacaagtttgtggacagtaccctgcgcaaagtcctccctggcctggtgagtggtcctgagcaagcttctatctcttcttggtgggactgctatggccagcgcatttgatcttgctgtgttgatagtggctacgaggagttcagggtctgtggctagctcaggaagaagaagcatagtgatcatctagtgatgtctgcacaggcataggtataggcagcacagcacgtgtgttcaacactgctcagatgaccagtccaaggatatgaatccttggTAATTTTGCACGCTGTGAAAGAATTTGGAGTCTAATCCAGACTCTGGTGTTGATAGTCCCATG

 

This mutation is a 1177 bp deletion beginning at Chromosome 2 position 153,903,913 bp and ending after 153,905,089 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
65992 Fluorophore-1 TCT TGA CCT CTC TAA TTT TGC ACG Quencher-1 MUT Probe
66822 GTG GTC CTC ACT GCT CTT CC Wild type Forward A
66823 TGA TGG CTT TGC TGA GAC AC Wild type Reverse A
66824 Fluorophore-2 TGA TCT CCC ATA ACC ATG TCT C Quencher-2 WT Probe
67402 TAG TAG ACT GGG CTA GAA TCT TAC TC Mutant Forward A
67403 GGA TTA GAC TCC AAA TTC TTT CAC Mutant Reverse A

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
66822 0.40 uM
66823 0.40 uM
67402 0.40 uM
67403 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.