Protocol 31805: Probe Assay - Gm4792<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=  105 bp

Wild Type = 105 bp

>chr10:94294873-94294977 105bp TTGCAACTGCTAGGCTCAG TGGCTCATGAAGCAAATTCA

Sequence

Sequence (deleted region in lower case)

AACAGGGTGACTCACTTCAAATGACGGCCGACAATGAATGATATACAATTAAGCGTGTGGGCCCCAGGGGCAGACTGCATGGACAGAGTCCTGGCTTTGTGATTTTGGGCAATTGAATCTTCTTGTGCCTCAGATTCTTCATTTGCAAACCAGGAACCCTAATGCTGCCTATCCTTGGAGACTAGGATGGGAAAGGAAGCCTGCTACATATGACTCAGCAGCAGAATTGTCACCCTGATGgtcatgggagtctgcttgcttggactaaacctttttatttctctccttattccaggactgcttgtgcccctcccccacacagcccagtggccaggattcctgcctaactatatcccaagaattgagcctaggactctctttggtctgctctggctcataaaagccaagacgaggtgaggcactcctttctgtttctgcttctccgttcactcaccaattcacaaatgattttctgcatgtgtgtttgtgtaatagagaactgtccctttaaatgtgtgtgtcacagatcttgtctcctgctgggattgcagtggcctgaaggcagggaacaaatactgccacaaaaggccttaggctttgtagtacacatggtctcagttgcaactgctaggctcagctgacaaagcatgaggtagccttagacaagatgagagatttcgtttacaaagagcaggcCACTGGCTGAATTTGCTTCATGAGCCATAGTTTCCTAACTTCCAAGCTAAAGCATATTTTAAAAAATTGTTTTTCATCTCTCATTCCCTGTCCGGCTTTTTAGAAATGATTGGGATTATGCTGTGTCTGTTTGCTTATTTTTAAAGCATAAGCAGGAGGCTTTTTGTGGATCTGTATTCGACTTTACTATATTGGCTATGCACGTTCCTCTAGCCTTTCTCCTGTGTTTGTG

456 bp deletion beginning at Chromosome 10 negative strand position 94,295,355 bp and ending after 94,294,900 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
37704 TGC TGC CTA TCC TTG GAG AC Mutant Forward A
37705 TGG CTC ATG AAG CAA ATT CA Common A
37706 TTG CAA CTG CTA GGC TCA G Wild type Forward A
37707 Fluorophore-1 TGG GAA AGG AAG CCT GC Quencher-1 MUT Probe
37708 Fluorophore-2 AAG CAT GAG GTA GCC TTA GAC AA Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
37704 0.40 uM
37705 0.40 uM
37706 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.