Protocol 36523: Probe Assay - Ddx31<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

>chr2:28845674+28845766 93bp TGGATTCCCTTGTCATTCAG CCCTCCCCCACATAACACTC

Mut= 99 bp

Wt= 93 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 CCTGGCTTCCTGGGTAACCTCACTTGATGGGTCACTCCCTAACAGCAGCATTGAGTCCAGACTTGGTTTGTTGGGCTTAAAGCAGACTTCTAATTTCTGGATAAGTGTGATGTTGCTATGGTGGCAGATGTCTCTGTGACCTTGGGGAGAGAGCTAAGCTACCTTCTTACTGTCAAAGACCTGCATGTTTTCAGAGAATAAAGGCAAAAAAGACTGTTTGAAGTGTGGTTTCTACAAGCAGATGGACATTTTTCTTTTCCCTTTTGTATAATGGCATGAGTAAGTTTTCTAGTTACTGTGAAAAGGGAGAGAAAAAAAACATTGGAagcctggattcccttgtcattcagtcacaattgaccttttttgtgtgtgttagctatgtaggattgtttctcagcctgagtgttatgtgggggagggggccttggatagcggagttattatgaggttgccttatatatcatcagtttgtaaaagcaaatcctgcagatactatagggtagagggcagactcaccccccagctgaaagtactggcttagtgggctttaatttaatcataagatgctgatgtcgctccagcattttgtctttatgtcattgaatatatttgcattaaagtaatatttatttcatattctcttttagatttctacaataaatacagtcttaaaaatgtccagtatgaccaggtaagaggttgaagctatttttgattttcttagggtttctttgagtacaagctgtccatggaaggcagagcacattcgtgtcgggtgctgggaacaactcaaactgctaaggtcttgacttagcgcttagcctatgggactccctacctgcttgtatgcagactggatttccttccatttcggcactcaagctccctactgcaagacatagctggtttaggacattaggacatctgccatctctgaaaatgacttgagccaaaatacAGTATTTGGTCGTGTTAGTTCTTAACAGTAAAAATAAGGTCAACCAAAATGATGGGAGTAAATATTTGCTAGGATTTAGAGGGATACTGAAAAGGTATTCTAAATGCTTGTTGCATTGGAAACACTTGTAAAAGAGAAATTCAGAATTAGACTATGAGCAGTGAGCTGTGTAGTTGTAGTTGACTTTGCATAAGCAAAATGCAAAAATATGGAAGAATAGAGAACCACCAGCCTTTTCCATTTATTTGCTTTTGTGACAGTCTCATAGTTCTGCTGGCCTCA

This mutation is a 635 bp deletion beginning at Chromosome 2 position 28,845,670 bp and ending after 28,846,304 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
49010 TGG ATT CCC TTG TCA TTC AG Wild type Forward A
49011 CCC TCC CCC ACA TAA CAC TC Wild type Reverse A
49012 TTC TAG TTA CTG TGA AAA GGG AGA G Mutant Forward A
49013 ACT CCC ATC ATT TTG GTT GA Mutant Reverse A
49014 Fluorophore-1 ACA ATT GAC CTT TTT TGT GTG TGT Quencher-1 WT Probe
49015 Fluorophore-2 CAT TGG AAG TAT TTG GTC GTG TT Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
49010 0.40 uM
49011 0.40 uM
49012 0.40 uM
49013 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.