Protocol 37646: Probe Assay - Sap30<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

>chr8:57487831-57487950 120bp CAGAGCGCAAGTTAGCTCCT CCTCCTTCTCAACCCCAGA

Mut = 105 bp

Wt = 120 bp

Fam = Mut

Hex = Wt

 

Sequence

MUT Sequence (junction in uppercase):

gaacagagcgcaagttagctcctcgatcaagaactaCCttccctaggtggatttgatttgcaattaagcattcttgtttttgttttgttttgtttctccgtgtaaccctggatgccct

This mutation is a 5368 bp deletion beginning at Chromosome 8 position 57,482,549 bp and ending after 57,487,916 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
51839 CAG AGC GCA AGT TAG CTC CT Common A
51840 CCT CCT TCT CAA CCC CAG A Wild type Reverse A
51841 GGG TTA CAC GGA GAA ACA AAA CA Mutant Reverse A
51842 Fluorophore-1 CAG AGG AGG GGC GTC TCC Quencher-1 WT Probe
51843 Fluorophore-2 ACC TTC CCT AGG TGG ATT TGA TTT G Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
51839 0.40 uM
51840 0.40 uM
51841 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.