Protocol 36222: Probe Assay - Dnai4<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

>chr4:103096477-103096601 125bp CAGTACACAAGGTGAAGTTCTTTTG GTGCCGGTATACTAGAATTGATAGA

Mut= 97 bp

Wt= 125 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case; and insertion with carrots ^g^ (T insertion):

 TGGCTCACAGCCATCTGTAATGGGATCTGATGCCCTCTTCTGGTGTGTCTGAAGAGATCAACAGTGAACTCACATAAATAAATAAATAAATAAATAAATAAATAAATAAATAAATCTTTAAAAAGTTTGCCATCATGCTGGCAGTTTCTATATGCATCTTGCCTCTTAATTACATTAACGGGCCATATCAAGTGACTGGCTATCCTATCTAGGTCTC^TTTCTGTAATGTACTGTATGTTATTCAAACAGTGACAAAAATATACTAACATCCTCAGATCGCATTGCCTCTgtatttactactaatacacaggcatgtgagcacaattttcaacagatatgtggctagttacatgtgaagtataacattttgaatcatttaagtttttgtgtttattttgtcctaagaccatgatacttagtatttttctgctgtactttttttgtttaccaagagttcgaaaccttgccctgtctagttattcactccatttaacaaccattgttttgcaggtgcttgattcaaaaggagttgatgtcacaccccgacctctctaccatccagatccacatgctgcgtcagcaaagccaaacaaactcttgacatcacaagaaggatcgcttggatcagattatatttcttcctatagcctctatcagaatacactaaaccctagcatgttagggcagtacacaaggtgaagttcttttgatttcactaattttgaacttccgtttggatTCATGCGTCCAGAATGTATATTAGTTTCCAGTTTTCTCACATCAATCTATCAATTCTAGTATACCGGCACTTACATTTTAATTATTCATGCTCAGAGCTTGATTAAATAGTTCCCATACATTCCAAAACAGGGTTTAAAGTTCACCTCTTGGTGAACAATTAATTGAAACCAGGAAAGTGAGAAGATGATAGAGTGAAGGATGCTCATTTGTTTACAATTTG

This mutation is a 450 bp deletion beginning at Chromosome 4 position 103,096,547 bp and ending after 103,096,996 bp (GRCm38/mm10). In addition, there is a single bp (T) insertion 73 bp before the deletion. 

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
48158 CAG TAC ACA AGG TGA AGT TCT TTT G Wild type Forward A Wt F
48159 GTG CCG GTA TAC TAG AAT TGA TAG A Common A Common
48160 CTA ACA TCC TCA GAT CGC AT Mutant Forward A Mut F
48161 Fluorophore-1 CAC TAA TTT TGA ACT TCC GTT TGG Quencher-1 WT Probe
48162 Fluorophore-2 CCT CTT CAT GCG TCC AGA AT Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
48158 0.40 uM
48159 0.40 uM
48160 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.