Protocol 31778: Probe Assay - Trf<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

>chr9:103228230-103228319 90bp TTCAGCTACCCCAGCATCTC TCCTGGGAACAGATGAATCC

Mutant=  106 bp

Wild Type = 90 bp

Sequence

Wt Sequence: TTTTGATGTGGACCTTCAGCCAAGGCAAAAGAAGAGCAGGTTGTCACCTTCAGCTACCCCAGCATCTCTCTAAGCAGCccagggttaggtgggaacatggcagaccgttggggcgaacggattcatctgttcccaggatcggagtgcagcagagccctgtgctctgtcatgactgccaccagggatgagaggagggtggaacagaggagaggtggccattaagacccttcgtaaagtggtgcgttctctccccaggcacggggagcgggtcctttatgcctccgccttctctcctgcagggctgtgtctggctgtccctgacaaaacggtcaaatggtgcgcagtgtcagagcacgagaataccaagtgcatcagcttccgtgaccacatgaagaccgtccttccgcctgatggcccccggcttgcctgtgtgaagaaaacctcctatccggattgcatcaaggccatttctgtaagtcctgctgccgggaacggtagcccctaTACGCCTCTGGTTTGACTCACTTGTGGAAAAAAAAAAATTGAACTGGGGGGTGGGGACTGTCAGGTGTCCAAGT

 

Deleted Sequence:

ccagggttaggtgggaacatggcagaccgttggggcgaacggattcatctgttcccaggatcggagtgcagcagagccctgtgctctgtcatgactgccaccagggatgagaggagggtggaacagaggagaggtggccattaagacccttcgtaaagtggtgcgttctctccccaggcacggggagcgggtcctttatgcctccgccttctctcctgcagggctgtgtctggctgtccctgacaaaacggtcaaatggtgcgcagtgtcagagcacgagaataccaagtgcatcagcttccgtgaccacatgaagaccgtccttccgcctgatggcccccggcttgcctgtgtgaagaaaacctcctatccggattgcatcaaggccatttctgtaagtcctgctgccgggaacggtagccccta

426 bp deletion beginning at Chromosome 9 negative strand position 103,228,289 bp and ending after 103,227,864 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
37632 TTC AGC TAC CCC AGC ATC TC Common A
37633 TCC TGG GAA CAG ATG AAT CC Wild type Reverse A
37634 ACT TGG ACA CCT GAC AGT CC Mutant Reverse A
37635 Fluorophore-1 AGG TGG GAA CAT GGC AGA C Quencher-1 WT Probe
37636 Fluorophore-2 CGC CTC TGG TTT GAC TCA CT Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
37632 0.40 uM
37633 0.40 uM
37634 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.