Protocol 35298: Probe Assay - Lurap1<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

>chr4:116144083-116144188 106bp CCTGGGTTGAAAGCAGAGTG TCCAAGAGATGTGCCTCCTAC

Mut= 104 bp

Wt= 106 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

GCAGCCGTGTTACCTCGATTGGGGCTACTTTTCATCCAGAAAATATTTATTAGCTTCCTGCTGCGTGTCTAGTTGGTGCCAACACAGGGAGAAGGCACTGAGCTTTCTTTGAGGTCTTCAGTTCCAAATCCAGGATCACCTAGTCGAACGCCTCCAGGGCTAGAAGTTGTgtgcataccactcggtacctcgctcccccaactgcgcggcttttctgagcgttacggcgccctcctcctgttcccccaccccaccccccgcgcggggcgggcggggatccacgggtttcgctccgcgttcgtagcagagaagggaggacccttgggaaccggaccctggtcgcccagtcctgttctggcttgggcccacatggaggggaccgcggagtcccagacgccggacttgcgagacgtggagggcaaggtgggcaggaagatccctgaaggactgctccgcgggcttcgaggcgagtgcgagctgggaacctctggggacgtgctgctcccgggggcgcccagcaccggccacggcctaggggacaagatcatggcgctgaggatggagctggtgagtgtgggtgctacaggccacggggaacgggatatttgtaggggcaggggcaggttcccggagttgggctggagaagagggtccttccaagatcaatccatacatcctgggttgaaagcagagtgaagtgacgtctcattatttgtctcccaagggtgaatgaaggGTTGATAGGTCCTGAGAACTGAGAGTAGGAGGCACATCTCTTGGAAGTTTTCCCTGCCCAATAATCGGAAGGAGAGAGGCGAGGGCTAAGTAGCACACAAAAAAAACTTAGTACAGCGCAAATTCTTCCCATCGGCATCCGGGGCCACCAATTTCTTCAGACTCCACTATAAGGCAACCG

This mutation is a 566 bp deletion beginning at Chromosome 4 position 116,144,128 bp and ending after 116,144,693 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
45721 CCT GGG TTG AAA GCA GAG TG Wild type Forward A
45722 TCC AAG AGA TGT GCC TCC TAC Common A
45723 AGG TCT TCA GTT CCA AAT CCA Mutant Forward A
45724 Fluorophore-1 TCT CCC AAG GGT GAA TGA AG Quencher-1 WT Probe
45725 Fluorophore-2 AGG GCT AGA AGT TGT GTT GAT AGG Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
45721 0.40 uM
45722 0.40 uM
45723 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.