Protocol 35485: Probe Assay - Depdc1a<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

>chr3:159515386+159515508 123bp TCAGTCCTATACACACATACACACA TTTCCTGAAGGGGGAAAAATG

Mut= 140 bp

Wt= 123 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 GACACTTTGTAACTCAACAGTATACTTTGATTTATACAATTACTAAGTATTAAAAACACTGCCTCAATAAAGAAACTGGAGATCCATCAAGAAAGACACCCAATGTCAACTTCAGTCCTATACACACATACACACACATACCAAACAAACAAATCTGTGTTAAAAGTGTTCAACattcctttcagtttgagtacaaactagaacaaaccaaagcatttttcccccttcaggaaaatacagaaaagataaatcatgaaagaataaccaatgaagatctagaaattgcacctgacaaccaagaaataagccaggaagatgttgaagaagtttggagatacgttattatgatatagtaagacaaaaataagcagtatttaagattctgaagtatgtggattattaacttgatttttttatccaaatatgcaaatgaataaaagtattacttttcaaaacttagaatttaaatatgcattatgatttttaataattgcttttggtacagatttagtacagtataaagaaagaagttataccttttaagtcatcactgtagtttgagatttttattggactcctctcaattttgttgagtaagtatatagtaaaagtaaatagtaaatataatccttgaattgttttaagagaaaaataaagtactcttatgtctttgtttgttgcaagtcatgttcttttcctcttattttataaagaaaaccattgtttatactctctttagcctgcaaaccattttaagtttgccatccatagaagaactcttaaacccaaaccaagtaattcctcaatatataatgtacaacatggccaacacaagtaagcatggagtggtcatactacaagacaaatcaggtaggtatttagaaaagaatctggaatcttttcaaacctagtaaccaaaggtgcATTTATAGGTTAGTATAGAAAGTAGAACATTTTCCTAGGACACATTCATTTAGCAATAATGATGGAATTTTTGGAAGTAAATATTATTTATGCTATCACAAAAAAGTATTTAATTGCTTAAATGAATTAATATGACTTTAAAAATATGGCAGTGATAAACA

This mutation is a 750 bp deletion beginning at Chromosome 3 position 159,515,449 bp and ending after 159,516,198 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
46187 TCA GTC CTA TAC ACA CAT ACA CAC A Common A
46188 TTT CCT GAA GGG GGA AAA ATG Wild type Reverse A
46189 CTT CCA AAA ATT CCA TCA TTA TTG C Mutant Reverse A
46190 Fluorophore-1 CCT TTC AGT TTG AGT ACA AAC TAG AAC A Quencher-1
46191 Fluorophore-2 AGA AAG TAG AAC ATT TTC CTA GGA CAC A Quencher-2

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
46187 0.40 uM
46188 0.40 uM
46189 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.