Protocol 37308: Probe Assay - Lysmd1<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

>chr3:95137426+95137520 95bp AAACCCTATAGCAGCCTCCA CTCAAACATTTCTGGCTTAGTATTCC

Mut = 107 bp

Wt = 95 bp

Fam = Mut

Hex = Wt

 

Sequence

Wt Sequence (deletions in lower case):

TTGGTAACTCAGTTAATAAAAGTGCTTGCCACACACACATAGTAAACCCTATAGCAGCCTCCAGGCTCTACCCccaagtacactgttttcaaccacacacaccaataagtttggaatactaagccagaaatgtttgagaacatctgctctacttagtgaaaaggaagaaaaagagcttgctatgttttatgaggttggtggattttactgtcagacattaatttcgttgcttttgtttgaagatggaacagattaagcgtacaaaccgcctttatactaacgactccatcttcctgaagaaaacgctctacatccccatcctatcagagcccagagacttgtttaatggtttggattctgaggaggaaaatgatggagaggaagaggtgcgcccgagtaaggatgagatcggatcatcttcggggaggaggaagaatcgagggtcagggtcgggtcggcccaatggcacaggcctcccgcctcaccaggaaacctccacccccagccatgacctttctgcctctgatttccttaagaagcttgactcacagatcagcctgtcaaagaaggctgctgcccagaagctgaggaaaggggagagtgggtgagtctctggccgctccttctagctccctgcagagaccctcttcttttagctggtcacaaactaagaagtaaccaaagtacCCGAGGACAGGCAAGTGGATGCCTTTCTCCCCATGTGGTCCCCAAATCATGAAAGAGCCAGTGGCATCTGGAATGGGGTAGTGGGTGGGAAGCAGGGTCTGTGGGGTCTTACGCCAGTGGTAGCGC

This mutation is a 614 bp deletion beginning at Chromosome 3 position 95,137,456 bp and ending after 95,138,069 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
51025 AAA CCC TAT AGC AGC CTC CA Common A
51026 CTC AAA CAT TTC TGG CTT AGT ATT CC Wild type Reverse A
51027 CCC ATT CCA GAT GCC ACT Mutant Reverse A
51028 Fluorophore-1 CAC TGT TTT CAA CCA CAC ACA CC Quencher-1 WT Probe
51029 Fluorophore-2 CTA CCC CCG AGG ACA GGC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
51025 0.40 uM
51026 0.40 uM
51027 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.