Protocol 35711: Probe Assay - Wdr73<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

 

>chr7:80897707-80897813 107bp TTCTGTCTACTACATGCAACCAAG GCTGGGGTGGTAAACTCAAC
Mut= 101 bp

Wt= 107 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 CTGCAGCATCTGTCTTTATCAGCATGCCCACTTCTCAGGATACAGACATAGGTGGGTGTACCTGGGGCATCTGAAGAAATTGTTAGCTTAGCAGACATTTTGACTAGTAGAGCCTACAAGTCCGTGCAGTTGCTCCAGGGGCCAAATGCAGACTGGAAAAGCCCTCTGAAGCTTGTCTGAAAGCTGACAGACGCCACAGAAGTTCAGgttttagtcggggttctgtgttcaggcttcctacttgtgacctttctaggtgaccatcatgacctgtggcagcctgggctaagtctgtggctttcttgagttgtgatcctgtccctaaagcaggatgagcctgctcatgtgttggacgagggcagctggtcccagggctcccaagtgggctagagaggaaaggctgctgtagggagggcaagcttccatgtctgctcttaagattccccgtctctctggaacaggttgctggtgaccagtggacttccagggtgttacatacaaatgtggcaagtcagagaggacaacggtgagtaaagttcacggggtggaaggtgaggttttaggaaagtgactcacacactcctgtctcttagcagagccaggaaaaaagcacttaagccaggggacagcacagtttctgtctactacatgcaaccaagatgagttagcatccatagtgtagcagacagGCCTACTAGGGCCTCCTTGAACTTCCTCCAGGAGTTGAGTTTACCACCCCAGCTGCTGGCAGAGCACCTAGAGAGCCAGGACCTCCCTGCTGGACGTGGGCAGAGGACGGGCTGAGCTAGAGCCACTGGGGCACTCAGCT

This mutation is a 482 bp deletion beginning at Chromosome 7 position 80,897,760 bp and ending after 80,898,241 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
46833 TTC TGT CTA CTA CAT GCA ACC AAG Wild type Forward A
46834 GCT GGG GTG GTA AAC TCA AC Common A
46835 AGC CCT CTG AAG CTT GTC TG Mutant Forward A
46836 Fluorophore-1 AGT TAG CAT CCA TAG TGT AGC AGA CA Quencher-1 WT Probe
46837 Fluorophore-2 ACA GAA GTT CAG GCC TAC TAG GG Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
46833 0.40 uM
46834 0.40 uM
46835 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.