Protocol 35265: Probe Assay - Hmgcs1<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

>chr13:119702361+119702480 120bp CAGGTGGCTCTTCTGTTGC TGACTTTGGGATAGGCTAGGA

Mut= 118 bp

Wt= 120 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 GCTAGTATGTTATGACTCATATTTGAAAATCAGTGATACAGTCATATATAGCTGAGTGTCTAAATCCACATTTCAAACTATTAAGAAATTTAATTAGACAGGATTTGGGGTCACTGTTTAAGGACACAAGCAGGTGGCTCTTCTGTTGCCACCCGCCctgctctctttgcagtgtagacataaacttgttttgtttcttaaaattaataagtaaataaagatacgagcatcctagcctatcccaaagtcaaggcacagtttctggcttctgtggatgccatcatttcttggtcttctctttcagagggaaaggataaagattttaccctgaatgattttggcttcatgatctttcactcaccatattgtaaactggtgcagaaatctctagctcggatgttcctgaatgactttcttaatgatcaaaacagagataaaaacagtatttacagtggactggaagcctttgggtaagaacgctgttatgatgtgcttatttctattttataaatatttacatatatatagacctagagatacagtgagaaaacattctattctagttgagtggttgtggtccacgcctttacttccagcactcgggaggcagaggcaggcagatctctgaatttgaggacagccagggttacacagagaaactccgtctcaaaaaaaccaaaaaactttatttaatgaaaaccagttCTAGAGAGGTAGTAAGTAAATGGCTTATCTACACAAAATCATTAGTCATTTCTACTTAAGATTCTGGTGTCTGTTTTTAGCTTCAGCCTCATTGTTAGCGCACAATACTGTAATCACAGCCGCACCAGTGTACACCAAGCTG

This mutation is a 548 bp deletion beginning at Chromosome 13 position 119,702,388 bp and ending after 119,702,935 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
45646 CAG GTG GCT CTT CTG TTG C Common A
45647 TGA CTT TGG GAT AGG CTA GGA Wild type Reverse A
45648 TGA GGC TGA AGC TAA AAA CAG Mutant Reverse A
45649 Fluorophore-1 TGC TCT CTT TGC AGT GTA GAC ATA A Quencher-1 WT Probe
45650 Fluorophore-2 CCC TAG AGA GGT AGT AAG TAA ATG GCT T Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
45646 0.40 uM
45647 0.40 uM
45648 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.