Protocol 42247: Probe Assay - Mob4<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=  110 bp

Wild Type = 128 bp

chr1:55148831+55148958 128bp TCCCAAAGTTTCTACCACTCC CAACTACACCCACTACATTCCAC

Sequence

Wt Sequence (deletions in lower case):

TGTATGTAATTGTACATAGAAAACACCTACATAATTTTTATATAACTAAAACATCTTTCATGTTTAATCATACATATCAACAACTTCCTATCCTTCAGTTGATCATAATCAGAAGTCTTACCACAtatacacggttaatgtgaacacctttgtaacataactgtatacagttctgaaaactgcattacaatagattctcaatcaaaaagtacttgtttttctagagtcttggtgaaatttctaagtactatccagaaaaattaaacagatttttattgtataaaaggataaccagcttgaccaccttaaaaaataaaagaagaaaatacacaaagcctggataataaatgtgctttttgcatttttaggcaattctgccttgaactaaatggacttgctgtcaaacttcaggtagtattttctttatgttaaagctgtaattcaaagttctcatttttaaaaatgattttgtatgcttagatttttccagtgtttcctattatcctttttatattttatatagaaatattataacccaaatgaccattttttccattatcattctacatattaaaaaaataaaattatttttaacagagtgaatgccatccagatacctgtactcagatgacagcaactgaacaatggatttttctttgtgcagctcataaaactccaaaagaggtgagaattgagagcttccagtacctgctcattggcaccattactttgggcctgaagtgaggccaaacatctcagatgggagcatttagggaagcaagtctgcttacatggcatccaagaaagagagtgaagagtcaggagcccagtgccctcaaagacactgcccatggcctgcttcctccaactcggcctcatctcccaaagtttctaccactcctgagagactggccagctgggacccacatcttcaggagataagcccTTGGGGAACATTCCATATCCATATATGAACAGAAATATTGTGGAATGTAGTGGGTGTAGTTGTACGATATTATACTTAACACTGCTGAATTTGTTTAATATCTGTGGGTGTGATGTATGTGCAGTTGTGTGTACATGTGTGTACATGCACCCGTGGAGATGAGAGAAGTCCATA

 

This is a 821 bp deletion beginning at Chromosome 1 position 55,187,235 bp and ending after 55,188,055 bp (GRCm39/mm39). 

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
60215 TCC CAA AGT TTC TAC CAC TCC Wild type Forward A
60216 CAA CTA CAC CCA CTA CAT TCC AC Common A
60217 CAA CAA CTT CCT ATC CTT CAG TTG Mutant Forward A
60218 Fluorophore-1 AGT CTT ACC ACA TTG GGG AAC ATT C Quencher-1 MUT Probe
60219 Fluorophore-2 AGC TGG GAC CCA CAT CTT CAG Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
60215 0.40 uM
60216 0.40 uM
60217 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.