Protocol 46017: Probe Assay - Kcnk12<em1(IMPC)J> Alternate2
Version 3.0

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

Mutant=  87 bp

Wild Type = 84 bp

Sequence

Wt Sequence (deletions in lower case):

GCTGTTATCTCCGCAGGTTTCGGCATGACAACACCAGCcacagtgggcgggaaggccttcctcatcgcctacggactgttcggctgcgcggggaccatcttgttcttcaacctctttctagagcgcatcatctcgctgctggccttcatcatgcgcgcctgccgggagcgccagctgcgccgcagtggcctgctgcccgccaccttccgccgcggctcggcgctgtcggaggccgacagcctggcgggctggaagccctcggtgtaccacgtgctgctcatcctgggcctgttcgccgtgctgctggcctgttgcgcctcggccatgtacaccagcgtggaaggctgggactacgtggactcgctctacttctgcttcgtcaccttcagcaccatcggcttcggggacctggtgagcagccaacacgccgcctaccggaaccaggggctctaccgcctgggcaacttcctcttcatcctgctcggcgtgtgctgcatctactcgctcttcaacgtcatctccatcctcatcaagcaggtgctcaactggatgctgcgcaagctgagctgccgctgctgcacgcgctgctgcccggctcccggcgcgcccctagcccggcgcaacgccatcaccccgggctcccggctgcgccgtcgcctggccgcgctgggtgccgaccccgcgactcgcgacagcgatgccgagggccgccgcctgtcgggcgagctcatctctatgcgcgacctcacggcgtccaacaaggtgtcgctggcgctgctgcagaagcagctgtccgagacggccaacggctacccgcgcagcgtgtgcgtcaacacgcgacagaacggcttctcgggcggcgtgggcgcgctgggcatcatgaacaaccggctggcagagaccagcgcCTCCAGGTAGAGCGGCCGCCCGCCCCCAGCGTCGCGGACCGGGCCGGGGCTGCGCGCCGCCGGGCGGGTTTGCTTCACTTCTCTCCGAGATGGCGCTTTCTTAA

This mutation is a 870 bp deletion beginning at Chromosome 17 position 87,745,950 bp and ending after 87,746,819 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
62729 GTT GAG CAC CTG CTT GAT GA Wild type Reverse A
67017 AAC TTC CTC TTC ATC CTG CTC Wild type Forward A
67018 Fluorophore-1 CGT GTG CTG CAT CTA CTC GCT Quencher-1 WT Probe
67019 CAC ACC ACA TGC GAC CAC Mutant Forward A
67020 GCT CTA CCT GGA GGC TGG T Mutant Reverse A
67021 Fluorophore-2 CCT GGC TGT TAT CTC CGC AG Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
62729 0.40 uM
67017 0.40 uM
67019 0.40 uM
67020 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.