Protocol 36007: Probe Assay - Wfdc17<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

>chr11:83703711+83703838 128bp ATGTCCAGGAAGCCACCAGT CTTGGCCCTTGTGTGAATGA

Mut= 128 bp

Wt= 128 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 CATCTGCAGGTAGGAGGGGCCCAGATCCACAGTGTTTCAGATTCTCATCTGTAGAGCTGTAAAGACACAAACCCCATCTTTCCCAGAAGAATGCTACATCATTATGTAACTGGATGTCCAGGAAGCCACCAGTGGGCCCTGACACTCCTTACTGGCTGTCTCTGCTCCAGctcttactgtgagacacagtgctacacagcaataaatatttcccttgctcatcattcacacaagggccaagatgtggtgtaacaactcacatggtaaggaaatgagtttgagactattttgtgattttgtttagcatctatcaaagaccaattagagagaagtcacttatgaaactggaaaaaggaacctggtgatgggcaggcactttctggaaagctcttgttttcatgcatttccccattgactgcttcctcccacacccataattctagctttactgaggctataaattatttcatgagttcagccgagccagagccaacatgaagacagccacagtcttgtttctggtggctttgatcactgtggggatgaacactacctatgtagtgtcttgccccaaaggtaagtgtgttaagggcatccaagctggaaagagaggggactctagaagatcactaggaccaggggatgcattattacagggaaagCCATGTGACAACTGCTTGGATCACACTCACTCCCTTTAGTCTATATAGAGTGATCATTCTGCATTCCTTAGCGTTCTTAAAGACAGAGGCTGGCAGGAGGGGGTTTAGTTTGAGAGAAACTTGCACAGAAATCAGGACAGCTCCGCTAGTTTAGAGATGTGCCTTGCATTAGAGTCCTGTGGGAGCACTTGCTCAGAGATGAA

This mutation is a 492 bp deletion beginning at Chromosome 11 position 83,703,768 bp and ending after 83,704,259 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
47559 ATG TCC AGG AAG CCA CCA GT Common A
47560 CTT GGC CCT TGT GTG AAT GA Wild type Reverse A
47561 CTA AGG AAT GCA GAA TGA TC Mutant Reverse A
47562 Fluorophore-1 ACA GCA ATA AAT ATT TCC CTT GCT C Quencher-1 WT Probe
47563 Fluorophore-2 TGC TCC AGC CAT GTG ACA Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
47559 0.40 uM
47560 0.40 uM
47561 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.