Protocol 32416: Probe Assay - Kcnv1<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

>chr15:45113088-45113234 147bp CCATCATTTTTGTGGCAGTG TCACACATAGGAAGCGCAGA

Mut= 154 bp

Wt= 147 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 

TGCTGTCCTGCCATTCCTATTGCTCCTTCATATATTTTTCTTTTATACACACAAATGTTACAGGTGGGAAATACATGTTCTTGTCACCAGAAAATATGTCACAAAAAATTAACTGATCTAAAGTTGCCTGCTAATGTATAAAACCTATTTGCATAAATTTCTAGTAAGAAAAAAGTGTCTTTTTAAAAGTCATAAGTAGGAAatctgtcagggaccaccatggacaaatatgaatatatattcctatattttgcacatatgaaagtgtgacctctctgtcaaatttaattctcttgtacgtctgtgttttttttttttttaattaatcacaaagttgtcttctttgaaagatacttcagaagaaaggagctgagtgaaactcttgactttaagaaggacacagatgaccaggaaagtcaacatgagagtgaacaggacttctcaaaaggaccttgtcccactgtccgacaaaagctctgggatattctggagaaacctggatcttccacagcagcccggatctttggagtaatctccatcatttttgtggcagtgtccatcgtcaacatggccctgatgtcagctgagcttagctggctcaacctacagctgctggagatcttggagtatgtgtgcatcagctggttcaccggggagttcgttctgcgcttcctatgtgtgaaagacaggtgccacttcctgaggaaggttccaaacatcatagacctccttgccatcttgcccttctatataactcttctggtggaaagcctgagcggcagccacaccacacaagagctggaaaatgtgggacgtctggtccaggtcttgaggctcctcagagctctgcgcatgctgaaactgggaaggcattctacaggtattcatatttcactcgtgccttttaatttcgcttttaaaataatctttagatatattggcacagaaccaccttacattcttactcaccctccaTCTATAAAGTACTAGGAAGAAAAAACTTCAAGATGCTATCATAAGCAAAGATATCTCCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCATATATATACACACATACTCTTTCTGTTCACAAGACAATTTGGACATAACAATTTGATG

This mutation is a 773 bp deletion beginning at Chromosome 15 position 45,112,795 bp and ending after 45,113,567 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
39053 CCA TCA TTT TTG TGG CAG TG Wild type Forward A
39054 TCA CAC ATA GGA AGC GCA GA Wild type Reverse A
39055 CTG ATC TAA AGT TGC CTG CTA ATG Mutant Forward A
39056 GAG AGA GGA GAT ATC TTT GCT TAT G Mutant Reverse A
39057 Fluorophore-1 CAT CGT CAA CAT GGC CCT Quencher-1 WT Probe
39058 Fluorophore-2 AGG AAG AAA AAA CTT CAA GAT GCT AT Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
39053 0.40 uM
39054 0.40 uM
39055 0.40 uM
39056 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.