Protocol 44057: Probe Assay - Mrpl39<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

>chr16:84732093-84732195 103bp GGTACCAACTTGTCTGTGTTTGG AAGTTATGTCAGTAGGAGTGGGACT

Mut= 137 bp

Wt= 103 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletions in lower case):

TGATTCTGGACGTATAAGCCGAATAAACCTTTCCCTCCTCAACTGACTTTTTGGTTATGGAGTTTTATGGCAGAAATAGAAGCCTAGACTCAGACAGATATATTTTCCTTTTCTTTTAAATTGTGGGTACATCAATCCATGGATTCACGTTGTTTCTCTTTAAATATTTGCTTATAGCTCTTTGAAGATTGTTTTGTTAATTGTAGATTAAATTCAGACTAGACTTAGCTTTAATAGATAttgttaaaggttttggtaattttgttaaaaatgactttgcatgtggatttttaaaaaaaaattcacatatatatgtatacacacacacacacacatatatatgtcataaatacataatatttctgcaaaagtaatccccagaattttgcttcaattgttctgtttcataatgtagtgcttaagcatgtaatctttcattgtggattttttcacatttctatcataaatcaattttccatccctagatctgagtgagtggtactgcagtaagtccatcctggctcttgtggacggacaaccgtgggacatgtataagcctctgaccaagtcttgtgagatcaaatttcttactttcaaagaccctgacccaaaagaagtgaataaggtattttcctgtctcctccatttttttcttcttctaaatcaacctatgtgcaatggagtatatagtctaaaaatacagacaaactcatatgtggtattctactaaatagtgttttactaggtatttttgtagttcaaaatactggtggtaccaacttgtctgtgtttggattgaccgcatgtcatttgtgctcataactttggataaccctctaaATCATCGGGAGTCCCACTCCTACTGACATAACTTGAATTTGTTCTGAAATCACCGTCTTCAGTGGATGGTTTTGGTAACTTTTTGAGTCTGTCCCACCCTCAACCTCCTGACTTTTACTGCTTAAAACACCACCTTTTATAGTTGTATTAAGTTTTATTACTGACGTTACGGGGTGTCTATATCTTTTATCTCTGAAGAGCCTGCCTTCGAGTTAAAGCTGCCATAGCT

 

This mutation is a 600 bp deletion beginning at Chromosome 16 position 84,732,127 bp and ending after 84,732,726 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
64671 GGT ACC AAC TTG TCT GTG TTT GG Wild type Forward A
64672 AAG TTA TGT CAG TAG GAG TGG GAC T Common A
64673 CAT GGA TTC ACG TTG TTT CTC Mutant Forward A
64674 Fluorophore-1 ACC GCA TGT CAT TTG TGC Quencher-1 WT Probe
64675 Fluorophore-2 TTG CTT ATA GCT CTT TGA AGA TTG TTT TG Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
64671 0.40 uM
64672 0.40 uM
64673 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.