Protocol 34611: Probe Assay - Plekhj1<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

>chr10:80795805-80795900 96bp CCTGAACCCTGGTTGGACAC GGGTTACCTACAACTCCCACT

Mut= 96 bp

Wt= 96 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 TTTAGGGTCTAACCTAATGACCTTCTATTAGCCACTTCCATTCCCAATAACCCAACTTCCCAGTGAGTCCCCTATTTGTGGGTTCAGGGGTCAGGATTCTGGAGGACAAAGCCAGCATGAGACAGGCAGAAGATGACTTGGTGTTAGAATAAGAGGTGTAGTTTGAAATCCTGgtgagaggaagctgggacttgtggccaacaggatgtagaccaactggacactgaccagaggtggctcttatcctttatccctttccaggcttcgtcgaagacctcagcaggaaatatcactttgaatgctgtagccaagagcagtgtcaggactggatggaagctctgcagcgggccaggtggggctttagggactgatgggagctgggtggaaacatgggggaatgtgaacagtgagcccaatcctttcctccagctacgaatacatgcgacagagcctcatcttctacaggaatgagatcaggaagatgactggcaaggtatgtggggcggtcagggatgggacagctttccctggccttgctcacagccaccgtgccctccacaggaccccctggagcagtttggcatctccgaggaggccaggttccagctgaatagcctgcctaaggatgggcggactacttgtattggctcccagttaaatgtgttggactgaccctctctgggcccctggctaccctgggaagtttttttttttttaatataggttttaatacatctgttcaaaaggtccagaggaggtgggcagggtctgtggtcctcgttctccatgccatctccctgccagcaccccttgagtgtccacatccagggaccgagcctggcacagctcccctatccattgccagatgaatgtataatgctagatgaagctgtgaaccccccccccccctcctgctgatagagcagggcatgggtgctggcctcagtgctcagcctgctctttaataaaggtacctcttttctgagccatgtcggaatgagtggagaagaggagggtgggacacaggctgtgggcacttgggaggcagggggaagaggagggtgggacacaggcagttccagagcatctgggacagagaacaaaacaggcaagctgggctggtggcacacacagtataggtgtaggcaggacttggggggacaagcctctggctcgcagtgtgcctgaaccctggttggacaccctcactgtggagatgtggatccatccttggGTTTTGGGGAGTGGTACTCATATAAGTGGGAGTTGTAGGTAACCCTGATGGGGAAAGGTGTGTTCTGCCCAAGTTGTAAGGGACACCTAAATCCTGGCTTTCTAAACAGGCTGTGTGCACTTGGGAGGCAGCGGGAAGAGGAGGGTAGGACACAGGCAGTTC

This mutation is a 1061 bp deletion beginning at Chromosome 10 position 80,795,850 bp and ending after 80,796,910 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
44015 CCT GAA CCC TGG TTG GAC AC Wild type Forward A
44016 GGG TTA CCT ACA ACT CCC ACT Common A
44017 CAG GCA GAA GAT GAC TTG GTG Mutant Forward A
44018 Fluorophore-1 CTG TGG AGA TGT GGA TCC ATC Quencher-1 WT Probe
44020 Fluorophore-2 TTT GAA ATC CTG GTT TTG GG Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
44015 0.40 uM
44016 0.40 uM
44017 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.