Protocol 46221: Probe Assay - Srfbp1<em1(IMPC)J> Alt 1
Version 2.0

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

>chr18:52483482+52483601 120bp AGGCTTTCTAGTATCATGGGAAT TACGAGCATCGGGAGTTAAA

Mutant=  153 bp

Wild Type = 120 bp

Sequence

Wt Sequence: aggctttctagtatcatgggaatacctttacTgtggcagggatttattttctagaacttagcttttgtattaagttttctaaagcaagacacaaaaagaatttaactcccgatgctcgta

Mut Sequence:  aggctttctagtatcatgggaatacctttacTGttttatgtcataaaaggatattgaatttgtattttttcaaaattttaatatatataaaatttatgtaaaatctatatacataatcatgttgaacagctgatgttttaaatactgatgcgg

A 4206 bp deletion beginning at Chromosome 18 position 52,483,514 bp and ending after 52,487,719 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
66143 TAC GAG CAT CGG GAG TTA AA Wild type Reverse A
66166 Fluorophore-1 CAG GGA TTT ATT TTC TAG AAC TTA GCT Quencher-1 WT Probe
67469 AGG CTT TCT AGT ATC ATG GGA AT Common A
67470 CCG CAT CAG TAT TTA AAA CAT CAG Mutant Reverse A
67471 Fluorophore-2 CCT TTA CTG TTT TAT GTC ATA AAA GGA TAT TG Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
66143 0.40 uM
67469 0.40 uM
67470 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.