Protocol 35513: Probe Assay - Ntmt2<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

>chr1:163704109-163704204 96bp ATAGGAAGGGTCAGCAAGCA GTAGCTCTGGGCTTCAAGGA

Mut= 98 bp

Wt= 96 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 TCTTCATAGAGTTCTTCGTGGTTCACTTCTTGGAGAAAATTACCTTGAATTGGAAGCCAAGATAATAGTTTGTGATTCTTGTTAAAGAGATAAATTTGGGAAAACCAAGCAGAACTATAAGTATTTGGCTTTGGGGGGGGAGGGGAATAATGAAATGTTCTCTCTATTGATGCAGTAGTAGATActtacctataaacacgtgtacaacaaatatgttgagtcacacacataaatgaaacattttatagtctaaaattgagccaaataaacttattttaaagaaataattgtaatgccgagactcgaggtgagtctctgtccttgaagtggcccacaaggtatttttctgcaggggccagggagagcgggaactggctgcgccttggactgcggttctggaataggaagggtcagcaagcacgtcttgttgcctgttttcagcagtgttgaattggtggacatgatggaatccttcctccttgaagcccagagctacttgcaggttaacgaggacaaagttgaaagttaccactgctacagcctgcaggagttcacgccccacttagggagatatgatgtcatctggattcagtgggtctcaggtcagtgttgcacctcatagcttccgcccttccccatttcctccttaggaaaggtgacacttgggactttcaacacgttttgctagctcaggctgtcatgtgaactcccaacaactctgggccctgaagttctcttaacattgctgcacaaacttggcaattcttacccctagggataGAGCTGGAGCCTGAAACAGCCTACATCTTTGCAGAAATGTAAATACTTGAAAACATTGGACCCATAAATGGTTAATCCTCATTATTTATTGGTAGAACTAAATGTTCTGAGACTTAAATCCTTAGGAAATGAAACAGCAATGAATTATATTCTCTCTCTCTTCCTCTCCCTCTTCCTTT

This mutation is a 596 bp deletion beginning at Chromosome 1 position 163,703,825 bp and ending after 163,704,420 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
46261 ATA GGA AGG GTC AGC AAG CA Wild type Forward A
46262 GTA GCT CTG GGC TTC AAG GA Wild type Reverse A
46263 GAA ATG TTC TCT CTA TTG ATG CAG T Mutant Forward A
46264 ATG GGT CCA ATG TTT TCA AG Mutant Reverse A
46265 Fluorophore-1 CTT GTT GCC TGT TTT CAG CA Quencher-1 WT Probe
46266 Fluorophore-2 ATA GAG CTG GAG CCT GAA ACA G Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
46261 0.40 uM
46262 0.40 uM
46263 0.40 uM
46264 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.