Protocol 35454: Probe Assay - Surf4<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

>chr2:26927006-26927115 110bp GGGAGCATCTAAAGCAGGTG AACAGGTCAGAGGAGACAAGC

Mut= 110 bp

Wt= 110 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 AGATAGTATTAGACATTGCCTATGGTCCTGTGCCAGATGTGTGTGACGCTGTCTTTGTCTCTGGGACTAAGTGTTGATCATCATGTGTCTAGACTTACGATGGGAGCATCTAAAGCAGGTGTTGAGTCCTGCAGTCTGCCCTGgcaaggcactggacctgctgagggcctctctgtcttccagagagctggcttgtctcctctgacctgttgtcatgatgaggttgcagccacctcaccagtgtgttctgtggtcttacagttccttcgagtcaccaagcagtacctgcctcatgtggcgcgcctctgcctgatcagcaccttcctggaggatggcatccgcatgtggttccagtggagtgagcagcgtgactatatcgacaccacctggagctgtggctacctgttggcctcatccttcgtgttcctcaacctgctgggacaattgagtaagtggctctctagcctctctagtatacttctggagcatcctgttcccttgactgatactccccattcattgttgttggccattgagaagctatgtgtgaacttgaggtctgggtaccccttcctgggctgaagGCTGATATGACAAACTTAAGAGCTTCTCAGCTGAGATTACAGCTCTGGGTGTTGAAATATGCCAGCCATGGTTTGGTTCTGAGCTTTTGGGTGGATGGTCCATACTCAGTTCCCTTGCACGCATTTCTTTAAACTTGCTCTAGAGATCGGAC

This mutation is a 441 bp deletion beginning at Chromosome 2 position 26,926,633 bp and ending after 26,927,073 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
46105 GGG AGC ATC TAA AGC AGG TG Common A
46106 AAC AGG TCA GAG GAG ACA AGC Wild type Reverse A
46107 TGG CTG GCA TAT TTC AAC AC Mutant Reverse A
46108 Fluorophore-1 CAC TGG ACC TGC TGA GGG Quencher-1 WT Probe
46109 Fluorophore-2 CCC TGG CTG ATA TGA CAA ACT TA Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
46105 0.40 uM
46106 0.40 uM
46107 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.