Protocol 32448: Probe Assay - Dguok<em1(IMPC)J>- Alternate 1
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=  96 bp

Wild Type = 129 bp

>chr6:83446426-83446554 129bp AGCAGGCCACAGAGGATATG CAAAGCCTGGATGGTTGG

Sequence

Wt Sequence (deleted regions in lower case)

 

TGAAAGTTCTGTTATATTCTCAACAGTTACTCCATACTCTGGGATTGTTGGCTCATTAAAACTGCAGCCTAAATCCTAAGGTCACGTCTGCTCACTGTGCTGTGGTAACTGGTTAGCTGTCCAAGCCTTCACCCAGCCCACAGTGAGTTGTACAGCTGCCAAGTTTTGAAGCTGGAAAAGCTGGCCCTGGCTCCCATGAGATGGGCGGCCGTGGCagaagttactggctgcctccctcacctggatttgggaagcatcccaacatgtgtcatttgcattgcagctgtgggcaaatccacctttgtgaagttactcatgaaaactcacccggagtggcaggtggcgacagaacctatagcagagtggcagaatattcaggctgctggagcccaaaaagtaagttttcagctgtggggggtagttggcagacatgcacgcagtctaattggagtaatttaatttactctgaagacatgaagtagcccagcttgtctcctttttaaaataacttcattctagaaagcaggccacagaggatatgacttgtttggatatgtttagttttgatttttgtggcttctgtatgtctgtgCAAgcgcctgcctcctgtctttgtggtttCCCAAGCATGCCCAACCATCCAGGCTTTGAAAATGAATGTTGTCATTCCTACCCTTAAGTATTTATATTGTTGTTATTGAAGCTGGCTGGCAGGAGCAATAGTCAACGAGGACATCTTAGATGTGGGAAGGGACCTCACTGAGCATCTGGTCACCCTTTGAGTCAATGCA

377 bp deletion retention of 3 bp (CAA)of endogenous sequence and further deletion of 26 bp, beginning at Chromosome 6 negative strand position 83,496,866 bp and ending after 83,496,461 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
38321 TGT ACA GCT GCC AAG TTT TGA Mutant Forward A
38322 CAA AGC CTG GAT GGT TGG Common A
38323 AGC AGG CCA CAG AGG ATA TG Wild type Forward A
38325 Fluorophore-1 CTG CCT CCT GTC TTT GTG GT Quencher-1 WT Probe
39135 Fluorophore-2 AAA AGC TGG CCC TGG CTC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
38321 0.40 uM
38322 0.40 uM
38323 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.