Protocol 31804: Probe Assay - Gm12695<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=  90 bp

Wild Type = 109 bp

>chr4:96769872-96769980 109bp ATGCTTAGACAGACAGCCATGT ATACACAACGCTGGCATGAG

Sequence

Sequence (deleted region in lower case)

ACTGGCAAAAGAGTTTGACTTTTGTCCATTTCTAGGTGCAAAAGCTTATTTGAGTTATAAATGCGCAATGTTCTCATGGTTAGGGTTCAATTTTGACTGTGTTCACTTCCTGTAGTAAGTGGTCTAGAGCAGATAATCAGATTATTAGGAAGTGAGAATAGAAAAAATAGTCTGAGAAGACAGTGGCATGGTGCTCTCAGGTACATGATGCTTAGACAGACAGCCATGTTTACCACTAAttacatctcacagggctgttcctgctgttcacgcttccatactcacaccttctgcttctcatgccagcgttgtgtatgatgaggagtgttccttatgactgacaaggtgcaaactctcttcacagtaagcagaatgagaagttgaagacagaacctcaaacctcactccagaaacccaggaatgattattcaagacgggtgagcagagacctgccaggacccactgactcttctgagcaacaaataactgctaacccagctgagaaggaagaaagcaagcaccaaagaagcagcctaaaacccgaaagcaatcaaaagttcttaaccagagtattcagtaacaggttcctggatggcagggtgagtaacaagagatggccaagcttttcttggtaactcattagaaaagtgcagaggagaacataatttcatgcatacatatacaaatagaaataagtactttttcatagactagaaattataaatgcttctttttcaacttgaaaataaattttgaggggattttgtgtagtacaattttgtctacaacaaattgagctggtcaacataactgtggcaaACTCACTGATACTCAGCAATGGATACAGTAATGAGCATCACTAGCCAAAGGTCTGAGGTTTATGTTATGTTACGTGATAAACGCCATGATCAAAAGCCACTTAGGGAGGAAGAAGGGGCTTATTTTTCAGACAGCTTATGGTCCATCTTGAAGGTAAATCATAGTATAGAAAGGCAAGAACCTTGAGTCCTGAACTGGAGCAGTGACCACTGAGGGATGGTGCTTATTGGTTTGCTCTCCGTGGCTTGCCCAGCCTGGTCTCTTATATAACTCAGGACCACCTGACCAAGGTAGCACCATTCCAAATGAGC

580 bp deletion beginning at Chromosome 4 negative strand position 96,769,948 bp and ending after 96,769,369 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
37699 ATG CTT AGA CAG ACA GCC ATG T Common A
37700 CCT CAG ACC TTT GGC TAG TGA Mutant Reverse A
37701 ATA CAC AAC GCT GGC ATG AG Wild type Reverse A
37702 Fluorophore-1 TCA GCA ATG GAT ACA GTA ATG AGC Quencher-1 MUT Probe
37703 Fluorophore-2 CAT CTC ACA GGG CTG TTC CT Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
37699 0.40 uM
37700 0.40 uM
37701 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.