Protocol 37510: Probe Assay - Kcna7<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

>chr7:45408742+45408832 91bp TCACTAGGCCCAGGAGACAC AACTCAAGACCCCAGCGACT

Mut = 99 bp

Wt = 91 bp

Fam = Mut

Hex = Wt

 

Sequence

Wt Sequence (deletions in lower case):

GACCTGTCCTGCCTCACGCTCCTGAGGGTGGAATGACAGGCTCATGTCACTAGGCCCAGGAGACACCACCTTTCCTGCCCctaggtctgaagaacagacattcagctatccaaaggcagtcgctggggtcttgagttgtgtagagcagtgaagtcacccctcaggaacactgacactgctattttcacattgaaacagtgactagaggttttggcctggctccctgacccctttctgtcccttctgcagttcctcgcccgactgaatggctccagtcccatgccaggagcccctccccgacagcccttcaacgatccattctttgtggtggagaccctgtgtatctgctggttctcctttgagctgctggtgcgtctggtggcctgccctagcaaagctgtgttcttcaagaatgtgatgaacctaattgacttcgtggccatcctgccttacttcgtggccctgggcacggagttagcccggcagcggggtgtgggccagccggctatgtccctggccatcctaagggtcatccgattggtgcgtgtcttccgcatcttcaagctctccaggcattcgaagggtctacagatcttgggtcagacactgcgggcttccatgcgtgagctaggtctcctcatcttcttcctcttcattggcgtggtcctcttttccagcgcagtctactttgctgaagtggaccgggtggacacccatttcaccagcatcccggagtccttttggtgggcagtggtcaccatgaccacggttggctatggggacatggcacccgtcaccgtgggtggcaagatcgtgggctctctgtgtgccattgcaggtgtgctcaccatctctctgcctgtgcctgtcattgtctctaactttagctacttttaccaccgggagacagagggcgaagaggcagggatgtacagccatgtggacacacagccctgcggtaccctggagggcaaggctaatggggggctggtggactctgaggtgcctgaactcctcccaccactctggccccctgcagggaaacacatggtgactgaggtgtgagggtcaactggggtctccagggagcagtggggtgggagggaggagggaaggcagggtcaggtgctgggttaaggactaagatggtaacaaaagggtacagattcttaatctgaaggcatgtcacatggtggctcctagggggaccttatgtgacaggaattgccaggatttgggttgtgtccagggctccccccATTGATTGCGCAATGTGGTAGAGCTGTGCAAATGTCCAGGGGCTCTATGGGTAGCACTGTAAGAGACTTGGCC

This mutation is a 1179 bp deletion beginning at Chromosome 7 position 45,408,776 bp and ending after 45,409,954 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
51520 TCA CTA GGC CCA GGA GAC AC Common A
51521 AAC TCA AGA CCC CAG CGA CT Wild type Reverse A
51522 CTC TTA CAG TGC TAC CCA TAG AGC Mutant Reverse A
51523 Fluorophore-1 AGG TCT GAA GAA CAG ACA TTC AGC Quencher-1 WT Probe
51524 Fluorophore-2 CCA TTG ATT GCG CAA TGT G Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
51520 0.40 uM
51521 0.40 uM
51522 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.