Protocol 36270: Probe Assay - Spa17<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

>chr9:37607544-37607682 139bp AGCTTTGATCCAGCAGAATG TTGGGGAGACCATAACAATTTC

Mut= 132 bp

Wt= 139 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 CATCTTACTTTTGGAGTTAGTAATTTTTCATGGTGGAAAGTTAAAGAAAACTATCTTTGGATAGAAATATTGCTTGTCTATATGAACTGTTTATATAATGTGTCTCTTTTGAAAACTCTCCCTTCTTTTTCACTTTTCCAAACTTCCGGATCCCTTAGTATATATTTTATATACTTTTTTACATTTGCCAGTGCACATTTAAGTCCATTTATTTAAAATACTGACATTTGTTCTGTGTCTTCCACTTTCTTATGAGTTTTATGAACACAGAAGTTATGAAGGTGTTTGGTGCTTTTGTAAATAAAACTAAGGGCTCATTCAGTGTTAGACTGATCATCATATTTTAATAATTACTTATGTCTACAGcactaggctatttagtgtcagcaagattttcaactgtcagtaaagaaactctgatttgaaacaagaaacttgtgtttctgcattgttcttggttcactttaatgaacacaaaataagtataaaattatttttgttatttagaaaccagctttgatccagcagaatggggggctaaggtagaggaccgcttctataacaaccacgcattcaaggtatagtcttatgaacctaccagatttagCTATATTTTTCTATCTATAAAAGAAATTGTTATGGTCTCCCCAAGTTTTGAGAAGTGTGTCAAGCATTAAATAACTTATTAAAAATTTTTATGATGAGAAATACTCTTCTAGAATATATGAATTGAGTACCAATACTTAGCATTAATTTAAAAATGACAACAGGAGAAAGGACTTGATCATCACGCAATTAGTCCCATCTCTTATTCTTTAAACTTGGGTATCATAAGTCATAAATTATATCTCACAAAATAGAACGCTATTAGTCCAATTTTTTTCATTTGCATGTTACAAAAAGACTAATTCAAATAGAAGATAATGATAGGGATTTTTTTTCTTCAAAGTTAATCTCTTTAGTAACTGA

This mutation is a 241 bp deletion beginning at Chromosome 9 position 37,607,588 bp and ending after 37,607,828 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
48249 AGC TTT GAT CCA GCA GAA TG Wild type Forward A Wt F
48250 TTG GGG AGA CCA TAA CAA TTT C Common A Common
48251 AAG GTG TTT GGT GCT TTT GT Mutant Forward A Mut F
48252 Fluorophore-1 AGG TAG AGG ACC GCT TCT ATA ACA AC Quencher-1 WT Probe
48253 Fluorophore-2 AGG GCT CAT TCA GTG TTA GAC TGA Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
48249 0.40 uM
48250 0.40 uM
48251 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.