Protocol 43924: Probe Assay - Mpnd<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

>chr17:56010921+56011030 110bp CCTGGGCTCCAGTTTGTACT CCTCCAACAGCAGTTCTTCC

Mut= 119 bp

Wt= 110 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletions in lower case):

TGGGCTTTGATCCTGAAGGAGGTGGGGGTCATGAATACTATTGGCAGAGGGAACTGCTGCAGGGAGACTGTCAGGGGAGAGACAGGGCTCCGAGACAGTCAGTCTCACCCGAAGTAGTACAGCCAGGAGAAGACAGGCTGAATCCCAAGCGTGGGGAGAATCCGCACCTCGGACACCTCTGCTCACCGTCGCCctctggcggttggtccaggcaatggcacctcgggctcaagcctgggctccagtttgtactgagtgaagctcccagctgatgttgttgctacactccattcccaccagagtcccaccagtgaaggggaggaggaagaactgctgttggaggaggaggaagaagatgtgctggctggggtctcatcagaggacaaaggccacagaccccctgggaagggttccttggagccaggtgaggggccagggcagaggcactatgatgtgggaagatggctttgatgtttcttaggcctggggaatcctgaatcctgattcatattcatattctctctctctctctcattctctctctctctctctctctctctctctctctctctttctgtctgtCTCTGTCTCTGTCTCTCTCTCTCTGAGACAGGTTCTCATTGTAGCCCTGACTGGCTCAGAACTCACAGAGATGCATCCTCTGAGTGCTAGGGTTAAGGGCATGTATTATCTATTTATTGTGATGGGGCATGTAGATCAGAAGTTGGGTCCCTTCTTCCACCATGTGGGGTCTAGGAATCAAACTCAGGTCATCACACTTGGTTGCAAGATCTGCTGAGCCATCTCGCTGGCCCTG

 

This mutation is a 389 bp deletion beginning at Chromosome 17 position 56,010,881 bp and ending after 56,011,269 bp (GRCm38/mm10). 

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
64346 CCT GGG CTC CAG TTT GTA CT Wild type Forward A
64347 CCT CCA ACA GCA GTT CTT CC Wild type Reverse A
64348 AAG ACA GGC TGA ATC CCA AG Mutant Forward A
64349 GCC AGT CAG GGC TAC AAT GA Mutant Reverse A
64350 Fluorophore-1 ACC AGA GTC CCA CCA GTG AAG Quencher-1 WT Probe
64351 Fluorophore-2 AGA ATC CGC ACC TCG GAC AC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
64346 0.40 uM
64347 0.40 uM
64348 0.40 uM
64349 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.