Protocol 34947: Probe Assay - 4930432K21Rik<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

>chr8:84159153+84159265 113bp TCATGCCTGTATTTTGGAAGC CCCATTTCCTTATGTGGCTATAC

Mut= 100 bp

Wt= 113 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 TTCTTGTTGAATTCCAGGGCCCAGTGAGAATCCTTGTCTCAAACAATAAGCTGAATGACCCCCTAAATATACTAATATGTTCACACAAAGTTCTTGAATCAGCCTCCAAAATGCAGGTCACCTGCTCATGCCTGTATTTTGGAAGCTGATGGCGGCAGGAAGTTCATAgccacagggtaagatcccatctcaatgaaaaagaaaaaaatctaattgtatagccacataaggaaatgggccctgcatcttgaaatgggctccttctcatcttcccatcactaggtgagcatcagtactggccgccttgccttcatcaaagacccactcacctagtccagctgtggtcctgttgtgctaagctaccagctatactaaacctgactacatccctgccccttacttccctctctaaatcaggggtcggcctccatccctcaaagccttctaagaacccacggctgagagactcccagagctccatgatggtccattcacactactccagagagtcggaagacagctcagaacctgctccttctgtagagctaggcggggaggaaccactacacgaagctttcaggtaggtggctcagaactctcatgcaggttcctccctcttcagtcattcacagcaagcccataggaaagacaaatgatccaggatgacctgccaccactgcaaccctttctgggaccctgGCCCAGGTCTTGTTTCTCATGGTTCTCTTTACTGGAGAAAGGTCACAGATAACATCAATTCACTCTTAGTCTCAACCACACTACTGGAAGAAGTTGGGAAGATAGAGCAAGTGCTGAGGAACCTGTTTCACAAGCACTAAGACCTGAGTTCCATCTCCAGAACCCATGT

This mutation is a 531 bp deletion beginning at Chromosome 8 position 84,159,196 bp and ending after 84,159,726 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
44873 TCA TGC CTG TAT TTT GGA AGC Common A
44874 CCC ATT TCC TTA TGT GGC TAT AC Wild type Reverse A
44875 TGA TGT TAT CTG TGA CCT TTC TCC Mutant Reverse A
44876 Fluorophore-1 CCA CAG GGT AAG ATC CCA TCT Quencher-1 WT Probe
44877 Fluorophore-2 AAG TTC ATA GCC CAG GTC TTG TT Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
44873 0.40 uM
44874 0.40 uM
44875 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.