Protocol 31561: Probe Assay - Cacng6<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

Mutant=  150 bp

Wild Type = 151 bp

>chr7:3424603+3424753 151bp GCTGCACAGGTCTTTCTGACT AAGCCGAGATTCTGCTGTTC

Sequence

Wt Sequence (deleted region is in lower case)

 

CTTGCATTTGGGGCTCAGCTTTTTCTCATTCCCGATCTAGTTGACACTGAGCCTACTACTTCCTTTGAAAGTCTAGGCCCTGCCCCCTGCCCCTTAGGACCCGCTGCACAGGTCTTTCTGACTCCTTGAGGCTCGCCTGAGCTCCAGCGGATCCCATCATCATGatgtggtctaacttcttcatgcaagaggaagaccgacgccggacagctgtgggccggcgtcgtgcccaagaacagcagaatctcggcttgactccggagcgagagggcaagatcaagctggggttgctggtggcaatcgtgggtgctacgctggctgtgctggctgtgggcaccgagttctgggtggaactcaatacatacaagaccaatggcagcgccgtctgtgaggccgcccatttggggctgtggaaggtgtgcatcaagcgactgtggcaggcggatgtgcccgcgggcagggagacctgtggcccAGCTGAGCTGCCAGGAGGTGAGAGGCTGTGTTCTGAGCCCAGATCTGTGTCCCACAGTCAGGGAGAAAGAAACTTTTGTCTGGGGGAAACTTTTGTCTTGTTCTTATCCCCCAAGAATGTTTGTGCCCAAATCAACCTGTGCTCACAGAAACTGGAGAGAAAGGGGGCGGGGAGAGTGAGAG

This mutation is a 311 bp internal deletion beginning at Chromosome 7 negative strand position 3,424,975 bp and ending after 3,424,665 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
37166 GCT GCA CAG GTC TTT CTG ACT Common A
37167 AAG CCG AGA TTC TGC TGT TC Wild type Reverse A
37168 TTC CCC CAG ACA AAA GTT TC Mutant Reverse A
37169 Fluorophore-1 TGG TCT AAC TTC TTC ATG CAA GAG Quencher-1 WT Probe
37170 Fluorophore-2 ATG AGC TGA GCT GCC AGG Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
37166 0.40 uM
37167 0.40 uM
37168 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.