Protocol 34646: Probe Assay - Faxc<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

>chr4:21936978+21937081 104bp TGTTCTTTGGTTGGAGGTTTG CCCACATTCATGGAGGACA

Mut= 108 bp

Wt= 104 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 CAAATACTCCTCTTGCCCATAGGCTGCACATAAGAAAGCAGGTTGGTTTGTAATGACTTCCCTGGGCCACCGGGTTCTAGTCATCTAAGAGTCCGTAAGACCCTCCCGCATGTCCACTATGCTTCTGGTCTCTAAACCCCGGATGAGGTAAGACCTATCACACACACTTTTGCTTGTTCATAGAGGGCaacaatcccacagggtcctcttgagaaccgaggcaacggaaaactaacaggtgggctatattctctgtttctgttttggttttgacaggaaacagcaagagctcgactccaaagatgctattattttgcatcagtttgcaagacctaacaatggtgtccccagcttgtctcctttctgtttgaaaatggaaacttacttaagaatggctgacctaccctatcaggtacttgtttgcaaatatgttttattcttgcgacatcgatatcacctagaggatggggtagttaatgtcacacaagtggatatcagagaaggcagggtctgaaggtaaccatacccagtcgtcagcaagagagacatgtgactccaggatctattgacttttgtttttccatcagttctaatgagggactcataatatttcttttaatgtgttctttggttggaggtttggctaggcttatggaacagctggtgagactgcctggtGGTACTTTTAGGTTTACATCTGTTATTTTGTCCTCCATGAATGTGGGGTTAGAAGGCATAATGGATTGGAAGTTCTAGGGCCTGAAGCTAGTGCCTCATGGCTCTCCCACCCAGCTATAGCTCTGGCCTTTGAAGAGATTTTAACTTCCCTATGTTCTGCTCAGGTGGTTTTTAAGGAAGTACCTG

This mutation is a 490 bp deletion beginning at Chromosome 4 position 21,936,545 bp and ending after 21,937,034 bp (GRCm38/mm10). In addition, there is a 7 bp insertion [TTTTAAG] at the deletion site. 

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
44126 TGT TCT TTG GTT GGA GGT TTG Wild type Forward A
44127 CCC ACA TTC ATG GAG GAC A Common A
44128 AAC CCC GGA TGA GGT AAG AC Mutant Forward A
44129 Fluorophore-1 CTA GGC TTA TGG AAC AGC TGG T Quencher-1 WT Probe
44141 Fluorophore-2 TGT TCA TAG AGG GCT TTT AAG GG Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
44126 0.40 uM
44127 0.40 uM
44128 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.