Protocol 32613: Probe Assay - Cep350<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

>chr1:155961315-155961401 87bp TCTCTGTTGTGGGGGATGTT ACAATGCTAGGTCAGGCTTCT

Mut= 87 bp

Wt= 87 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletions in lower case):

 

CTTTATAGCAAGACCCTACCTTATTTTACCTTATTTATTCTCTGTCTCTGTCTCTGTCTCTGTCTCTGTCTCTGTCTCTGTCTCTGTCTCTGTCTCTCTGTTGTGGGGGATGTTCAGGCCATCGgtgtgaactgagtatccttcttttataaatggcttgagaagcctgacctagcattgtaagtgcattggataagggctgaagtggttctcaagcctttgatcatgtttgggagcacatgaaactctatagtttctgaagttaacttcagatctctgactctattactatcactttttctcagtagatgtaaccacatcatgggatgcactttctcagaccaaggctgctgtaagtagttttagtttctattttaaacatttacttggtttaaaattgatactatgttatgatgtgattaattagaatatttaattggaaaacaaaggcatagattacacagtgaacaaatgtgagtagtgaagaagtaaactagacactgcagtagggtctcgGGTCTGCTACTGTCATGACATGTCAAGTATTGGAGTGACCTGTGTGGCTCCTCACCTTTAATTAGCTGGTTCACTTTGTGAGTTGATAggtaGGGTGGATATGGGGAGGTTGGATGGTACCTGTGTAGAAGGCTCATTATCAGGG

This mutation is a 392 bp deletion beginning at Chromosome 1 position 155,960,980 bp and ending after 155,961,371 bp (GRCm38/mm10). In addition there is a small indel 1 bp (T) insertion and 4 bp (GGTA) deletion 88 bp after the 392 bp deletion that will not alter the results of the deletion. 

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
39560 TCT CTG TTG TGG GGG ATG TT Common A
39561 ACA ATG CTA GGT CAG GCT TCT Wild type Reverse A
39562 AGG TGA GGA GCC ACA CAG GT Mutant Reverse A
39563 Fluorophore-1 CTG AGT ATC CTT CTT TTA TAA ATG GCT T Quencher-1 WT Probe
39564 Fluorophore-2 CCA TCG GGT CTG CTA CTG TC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
39560 0.40 uM
39561 0.40 uM
39562 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.