Protocol 32145: Probe Assay - Myom2<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

>chr8:15069061+15069141 81bp ATGCCTGCTCCTGAAGGACT CCCAACATACATCCCTTGCT

Mut= 82 bp

Wt= 81 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence: (deleted region in lower case)

 

TCGTTGTCTGATACCACTCTCACCAGTCCTAAGTGTTAGCACAGGCACCTACCATTCCTTGAAGACATCAGTCCATTCTTGTATATATTGGTTCTTCACTTTGACAGGAAGGGATGGCTCTAATCCCCCAGTTGTAAGGCTGACACTTCTTCTCTGTGATCTCTCCACCTCTACATTCAGGGATCCTCTCCAGGGCCCCAAACATGCCTGCTCCTGAAGGACTCTAAGGAgtgcccaagtatttcagtctctcatgccaaagaaagcaagggatgtatgttgggcatagctctattaataaaccacagccgcctgctgttctttgtagctttctgtgtatactgacacatactgaaaagaggagcgcacaagtagaaaacgttgtttggcttttccccatcatcttttatcccttcccactgagggatgggtacacagagggggcctgtctagtctgtcagtgtcaccaaatctaccagttgtcctatgaccaacggagcttcttggctctcttaggtaccggtctcaagcggcttcttacggagaagctaagaggcagcgtttcctcagtgagctggcccagctggaggagaatgtccacctggcccggtcacaggctcgggacaagctagacaagtacttcatggagcagacggtaggaggacctgcaagctcctccctcggggtgtagaaaaggagagtttgtctgcacgcaaatggtctaatgcttcactaaccGTGGGCTGCTCCTGCCACACCCACTCTGACCACCCTCTCTTCTCCCCCATAGGTGGAAGACAATCTGGCGTGGGAGAGACACTCATTTGAAGACCGAATGAGCAGGGCCCCTGAGATCCTGGTGAGGTTAAGGTCCCACACCATCTGGGAGCGGATGTCTGTGAGGCTCTGTTTCACTGTGCAAGGATTCCCCACGCCTGTGGTGCAAT

This mutation is a 508 bp deletion beginning at Chromosome 8 negative strand position 15,069,595 bp and ending after 15,069,088 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
38450 ATG CCT GCT CCT GAA GGA CT Common A
38451 CCC AAC ATA CAT CCC TTG CT Wild type Reverse A
38452 CAC CTA TGG GGG AGA AGA GA Mutant Reverse A
38453 Fluorophore-1 TAT TTC AGT CTC TCA TGC CAA AGA Quencher-1 WT Probe
38454 Fluorophore-2 TAA GGA GTG GGC TGC TCC T Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
38450 0.40 uM
38451 0.40 uM
38452 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.