Protocol 35987: Probe Assay - Trmt10b<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

>chr4:45301038+45301195 158bp CCACCAGTGAAAATAAAATAGC GAGCACCAGAAAGGAAAATGA

Mut= 148 bp

Wt= 158 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 TGGTGGGGTGAGGGTGGTGTTCTCTCTGATTACAACCTGGAGAGGGATATTATTCAGAGAGAACTTTTGCCTTGCGTACTCAAGGCCCTGGGTCAGATCCCAAGCGATGCACACAGTTATAATAATTCTGTAATGTTCACGGTGAATACCCACCAGTGAAAATAAAATAGCAAACCATCAtagtggtaaaattaaaaataaacacaagccagttgcaggcttgttttaaagacaaaaaccttttcttctttaactttatatataaatgaaaaaaggtggcgggcattcattttcctttctggtgctcatgaagttctcaaacaattttgttttctggataatggaagaaaaacatgcaaagaaaacagagacactgggaaaggatagtctcctcaaagaagagcaaaaggaagcaggaaagagaacgaaggaaagccaagcgtgcagaagaccccggtaacggtgagcgtggttgccggtgaaaggtcatggctttcagcaaacttgtttttatgttgctgggagagtagggcctattactcagttcctctgcaagtcagcggaccagtgTCAACTTCGAGGTTGCTTTTAAAGTAGAAGCTAAGTGACCATGTGCAGCTGTAATCTCAGAAACGGGATGACGCCGGCGGTAGCGCATGCTCACTCATGCTCTGTGCTAGCTTAAAATCATGCATTGAGTACCTCAGGGCTGCTACGTACTGAGATTATCCGCTTTGTGTAGA

This mutation is a 390 bp deletion beginning at Chromosome 4 position 45,301,069 bp and ending after 45,301,458 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
47481 CCA CCA GTG AAA ATA AAA TAG C Common A
47482 GAG CAC CAG AAA GGA AAA TGA Wild type Reverse A
47483 TTT TAA GCT AGC ACA GAG CAT GA Mutant Reverse A
47484 Fluorophore-1 ACA CAA GCC AGT TGC AGG C Quencher-1 WT Probe
47485 Fluorophore-2 CTC AGA AAC GGG ATG ACG C Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
47481 0.40 uM
47482 0.40 uM
47483 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.