Protocol 45342: Probe Assay - Trim10<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=  100 bp

Wild Type = 87 bp

>chr17:36872134+36872220 87bp CTGAGGAGCAGGCAGGAGTA AAATTCCCACTTCCCTCTTGA

Sequence

Wt Sequence (deletions in lower case):

CTGAGGAGCAGGCAGGAGTAGGGGGCTCTGATTTTACTgcctaaccttgggaggtgtgggcatagatcaagagggaagtgggaatttataaggaaagggaagaaaggacagatgagatgccacaggaagagggggaaaagatgggagaagatctcgctgtgaggagagtaaaatgttctccctggctctcatcttcccttctcagactcaggtggccaccaagaggcaacaggtgatctcacagtttgcgcatctgagccagttcctgcagcaacagcagactgccctcttagcacaactggaggggttagacggggacatcttgaagcagcaggaagagtttgattccctggccactggggagatctgccggttcagcaccctgattgaggagctggaggagaagaacaagaggacagccagggggctcctgacggtgaggcctggcctccagggtctctggaattactgcctaattggtacctccttgacatacatgtttaggcactgaagatggaggaggtacatggggaaagggggttggattgtaatctgtgcttccccaagagctgcctggtcactgtgctctctgttgttggtctctccttcctgccctccgtagaatggagaggaaatgaggatatgacagtaaaaggaggtggaagttgctcctgagacgtatcacacactgtgcaaacaagtgccaggtgttgcctccaaagcaaaactttcccctccatggccaggaagttcttcctagactgtaccacttccttgaactggaggagagttattggggttccaggagcatgctttagggagatgcactgggtactgggaaataagagactttggtgtagaacttgctcacttttgcctatagctggggagaagtaggtcgtatcatcaactctgtccatgaaacaaaggtctgggttctggtcttaatgtccaaggctgggaatacggcagtattttgggttttatggtcataacaaaagtgggagagaaaaactagaacctaattctctctctctctctctctctctctctctctctctctctctctctctctctctctgtccatctgttcctccctctctccctccctcctttcctttcttcctttcccctctcaggatatcagaagcactctaataaggttagtgataaagtatctacttttttcttccccatagacattcatccatacaccacgtacctagttattgacgtgaaaataaaccactttctcagtttagggtacagtcatatataaatgtacaaatgctcaaatgtgttctcagatagtgctgtagatctacccacttgtgcagcagtggggtccatctgagcggatccaagtctctatgtgggaagttgctcctcaagGCCTTGTTAGAAAAAGATGGCTGTCTGGGGACATCATTAGAAGACAATAATTCTGGGGCTGG

 

This mutation is a 1354 bp deletion beginning at Chromosome 17 position 36,872,172 bp and ending after 36,873,525 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
65358 CTG AGG AGC AGG CAG GAG TA Common A
65359 AAA TTC CCA CTT CCC TCT TGA Wild type Reverse A
65360 CCA GCC CCA GAA TTA TTG TC Mutant Reverse A
65361 Fluorophore-1 CTA ACC TTG GGA GGT GTG G Quencher-1 WT Probe
65362 Fluorophore-2 CTC TGA TTT TAC TGC CTT GTT AGA AA Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
65358 0.40 uM
65359 0.40 uM
65360 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.