Protocol 34822: Probe Assay - Myh13<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

>chr11:67329447+67329555 109bp TCAGATGACTTGGGGGTGA ATGAGCATGGGTTTAGAATGC

Mut= 98 bp

Wt= 109 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

GTTAAACCAAGACAACGTCCCTTGACTTTAGCTATCCTAAGATCTATCTGGCAATTCTGAAGGCCAACTACTCTTTAGCTTTACATACCAGATATTCCTTCTGGGCACACACAATGCCAGGCTGGTCTGGGAGGCACCAGACTAGAATcaggttaaccaaggggagttattgctaggtcatctccaggcacagaaatgatccattaagttgtggcaacgccctcactctgctgcagtatctcactatgccgcgcctaccccaggagagaacaggtcagtgacgctgcaccctcgccttgcttttcctcgttctacccaggagctcactctgaacagtgaccaagtcttccccatgaacccccctaagtttgacaagatcgaggacatggccatgatgacccacctgcacgagcccgctgtgctgtacaatctcaaagagcgttatgcagcctggatgatctatgtgagagccttttgacctctttctattttgctgcctctctgaggaaaaaacaagatccttaatcttttgaatttcccacagacctactcaggcctcttctgtgtcaccgtcaacccctacaagtggctgccggtgtacaaccctgaggtggtggcggcctaccgaggcaaaaagcgccaggaggccccaccccacatcttctccatctctgacaatgcctaccagttcatgctaacaggtaagtgaagcctttgatttgtatgattgtaaggcattattggcctagtgtttgggcccttatataccccacacatctaaacaatacagtagcagccagcatgaacttgggagcctcagatgacttgggggtgaatattccaagccatggcacttttattatagtagtcatggtgaagggcaaagCCAGGGGTTCTTTCTGAAGCATTCTAAACCCATGCTCATTTCTGGGGGTGGGGGTGCATTGTAGAACACAAGGACACTGCTCTTCTATATTCCCAACTTACCCTGCTATGGGTACATTTAGACGAGACTCCCCAAA

This mutation is a 738 bp deletion beginning at Chromosome 11 position 67,328,779 bp and ending after 67,329,516 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
44572 TCA GAT GAC TTG GGG GTG A Wild type Forward A
44573 ATG AGC ATG GGT TTA GAA TGC Common A
44574 AGA TAT TCC TTC TGG GCA CAC Mutant Forward A
44575 Fluorophore-1 AGT AGT CAT GGT GAA GGG CAA A Quencher-1 WT Probe
44576 Fluorophore-2 CTG GTC TGG GAG GCA CC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
44572 0.40 uM
44573 0.40 uM
44574 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.