Protocol 43930: Probe Assay - Mitd1<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

>chr1:37882850-37882964 115bp GGCATGTGATAGCTTTTGAGTC CATGGGACCAAGTGCCTCT

Mut= 98 bp

Wt= 115 bp

Fam=Mut

Hex=Wt

The WT Reverse primer (primer 64370) anneals over the nucleotide sequence containing mouse genomic variations rs585271614.

Sequence

Wt Sequence (deletions in lower case):

AACAAGAAAAAATTTCAATTTCAAGAAAAAAATTTAAATTTAAAAAAAGGATAGAGCCCTTTCTTAGCTTCTGCGAAGGCAGGAATCGCCTTTGGAATAGTATTTCATCTTCTTTCCCTATGGGTATGAAACATTTTGCAATGTGCAGCCTAGAGTAGGCATGTGATAGCTTTTGAGTCAGTAAGTTTAAAAGATTGTAACTTATCCTGATTgtcccctgtgtgccatataactgtgtataaagaaagtgctcagaggcacttggtcccatgggtagaaccacctgccaacaaaggtaaagaccagagttcaatccctgggaacccacatacaccccccccccccccacacacacacaaaagattagagatgtaactttttaaagtggtccctggttacgttactgtagcttgtgttttgcagatggaaaatatcacaagcaaattaaaatagaagagaatgcaactggcttcagttacgagtcactctttagagaatacctacacgagacagttacagaagtttggatagaagacccttatattagacaaactcatcaggtaagtttactaagatagaatgaaatgtgactttaaatgtacagtcagctacctgtgtttatgggtttcctacccacggattcagtggaggatcagggatataggtaaaaattccatctgtactgaacatatacaaacataacaagacaagggtaacgaatggtgtttacattgtgttaggtatcatgtagtctagaggcgatttaaagggcccaggaggaggtatgtgggttttgggatgatactatattgcttaaaataagggatggaaccatgtgtggatatcttagtatctggctaagggagggtatcctagaatcagttctcttaaatagttttgttagaccttggagtttccttcaggatacagtgcttataaagtacagtgttttattttgctccctgtgcacagtgcccagtaatttgctgtagaagcttaaatttaaaacttccatgtagcttgctatatttgggtccatcatgtgtttccctgtggaacagcgcaccatctttactcaaattattttaatccacctctttggtggtgagccctgaagaagcaccaccagatgggggagctcctccaccagggaggcccccaggcattcctccaggcgtgccaacagctctgctacagcttggtaacgatgggactgcaggcgttcagattcttccttctccgcagcctggttcggatccggctggctgaggattatagactcagttcttgccggagcactgtgaagctaggactaatccaccctaatgcctttcagctctataacttccttcggttttgtgagatgctgattaagaagccgtgtaaagtaagaaccattcaccttctcacctctgggtatgaagtaggtatctgaaatgcctcattctttcttaacttactgtgtccgtcatatgtgacctgatcctacactgtgtgccaagcatagactgtcttaagaaaaaaagctgttgatttgagagtgttgacctcgttcgcattcagccattttgctgagctctttgaatttgagtttgtcagggtggtctcagtggccctgcagtctgcaccagcgactcttctgttctttactccatgtgctgggtttcgactgtggctgtgacttccttgatttagacttgagtagaagtgtatctgaagaattatactttccagggttagtctaagcagtaggttgtctcacacggataactgagcatccttgctaggatacgtttaagaagctgagcatggtgccatgtgcctgtgctcttagcaacagggaggctgacgtaggagggagttgtgtgatagtctcaattgtttaattgtttatataaaaacaatataaaaagaaaagaattacccaaccaacagttgcttctttgagttagcaatattgaaattgatataaatcttttcagtttaatctctctgtagatttatttatttattgtatagctcatggatagttaataacagaggcagtaaattatgttttcatgctatcaatagggtttggggaacacacagcaaagtagtggcctggaagaaataaagcagtccctcgggagtcacggagtggtgctggaaataaactactcttcatctatacatgaccgagaaattaggttagtgtataatgttttactttttttgtagctgagatatactttaaaaattctaatgtaatataattaaatacatgtatttaaatattcaaatcaagctcaaatttagtaattgtatattggacaaaaacaaacaaacaaaaaaaacccatcttggtttacctcttagagtgtgttaattcttagtcaagtaatatttacaggtcctatgtcatGTGGGAAGGAGTTCTGAGTTCTGTGCAGACCATAGCCACTGTTATGTTAGGAGATAATTCCTTTCCTACTGAAATTAGTTTACCTGGTGTGGCTTTGGTGGTATGTTCTAGTAGTGTACTGTCGATGAGAAAATGCATGCACAGTTTCTTAGTGATCCTTTAGCATAGTAGATTTCATATGCTACTGCAAAGAGACCCTGTTTCTAATAAAAAGTAAATCAGAAGCTAAATATGTAAAGTGG

 

This mutation is a 2192 bp deletion beginning at Chromosome 1 position 37,880,718 bp and ending after 37,882,909 bp (GRCm38/mm10). 

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
64369 GGC ATG TGA TAG CTT TTG AGT C Common A
64370 CAT GGG ACC AAG TGC CTC T Wild type Reverse A
64371 AAC AGT GGC TAT GGT CTG CAC Mutant Reverse A
64372 Fluorophore-1 CCC TGT GTG CCA TAT AAC TGT GTA Quencher-1 WT Probe
64373 Fluorophore-2 CTT ATC CTG ATT GTG GGA AGG AGT TC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
64369 0.40 uM
64370 0.40 uM
64371 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.