Protocol 36049: Probe Assay - Pus7<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

>chr5:23776133-23776223 91bp CCCTAAATGTTGATGGAC TGGAGCACATGCACTTCTATAC

Mut= 90 bp

Wt= 91 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 TCAAAACCTGGGACTCACCAGTGCTTATATAGACTGGCAGGCCTGAGGCGCCAGGATCCTCTCCTCTCTGCCTCCCCAGCATAACCCAGGCCTGTGTTACCACACCTGACTTTTTCACCTCAAACGCAAGTCCTCATCCCTGCTTGCTCCTCAAGCACTGTACTGACGGGGCCCTAAATGTTGATGGACTTACTTTTGCGTTGGTTGatatgcgctatggtaatattttagaagctcctagtatagaagtgcatgtgctccattaaaataagagaagattgcttggtattatttgccaataaagattttccttaaatctcttgttttgaacaatggtgaactttgtttactttttactaatagtaaaattaggtagaaagtaaaacaacatccacttcttgaaagctttggttttcttttcatcagatactctgactttgtcgttcacgaaatcgggaaagatggacggatcagccatttggatgacttatctgttccggtggatgaagaggtaaaaatgtctctagatcaagctctcttctgtatgtagtcactcttcctaatgatcctgGACAGGACGTGTTACTGTCTGTGTGTGACTCTCTTGCTTTCTTGTGGTTGAAGGACCCTCCAGAAGACGCTCTCACTGTCTTGACAGCTGAGGACAGGCAGCAGTTGGAGGAGCTCCAGCTTTTCAAAAATAAAGAAACCAGTGTTGCCATCGAGGTAAGCGGCGCAGGAAGTGACGTCTCCGGGCTGGGCTGCTTCTCATTGGGTG

This mutation is a 364 bp deletion beginning at Chromosome 5 position 23,775,824 bp and ending after 23,776,187 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
47706 CCC TAA ATG TTG ATG GAC Common A
47707 TGG AGC ACA TGC ACT TCT ATA C Wild type Reverse A
47708 CCT TCA ACC ACA AGA AAG CA Mutant Reverse A
47709 Fluorophore-1 ATG CGC TAT GGT AAT ATT TTA GAA GC Quencher-1 WT Probe
47710 Fluorophore-2 TGG TTG GAC AGG ACG TGT TAC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
47706 0.40 uM
47707 0.40 uM
47708 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.