Protocol 38016: Probe Assay - Ptpn18<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

>chr1:34473174+34473298 125bp TAACCCAAGGCCCCAAAGA TGCTTTTAAGAGTTCCAGGGAC

Mut= 140 bp

Wt= 125 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case and insertions with carrots ^g^ (GCACCCATTGCA insertion) ):

GGTAGAGACAAGTGAACCCATTGAGGGGTTGCCAGTCAGTCAGGCAAGCAAATCTGTGAGCAGCCTGTTTAGTAAGAGATCCTTTCTTAAATTTAATATTGACCTTTGGTCTCCACACTGTCAACACACACACACACACACACACACAAAATGGGTGCATACTAATGTACAAAAAACCCCAAAAGGTTTTATTTAAAGTTTTAATCTAGGGATCCTGCAATT^taagaggtactgatctccttcaggtcagctcagaaggggttgtggtgtctgtacagttaggcagtcctgacatcccagcacttaggaagctgaagtcagaggctagcctgggcaacctagcacaagacctgtgggaaaaggaggaggaacagaatggggaaaggggcattgtgggtaggctatgatggtgtctgctctgattcacgattgtcagtgctggctgcggacgaacaggtgtcctgtgcgctgttgactatgtgaggcagttgctgctgacccaggtatgacgtcctctgctctgtgtgcagtatgtcccatgtctgcctaccagggtctggtcacacttcccctctctatcccagacaatccctcccaacttcagtctcttccaagtggtcctggagatgcggaaacagcggcctgcagcagtgcagacagaggtatgtgcttgccccgtcttcctgttcctaaggacgagggaggtttgcttagcttcccaatctccccttctcaatgaccactcaccccaacaggagcagtacaggttcctgtaccacacagtggctcagctattctcccgcactctccaggacaccagcccccactaccagaacctcaaggaggtacagagcccccaatttcaccctttctgccagccatcaacacctatacccttcttgggaacagcaaccgacttactgtgaaaggcacctttgcctcctcagagctccttccccaggctgttccctaagctcacctaggcacccacagtgacctgcagcagggctatggcctcaggaccgaccctgcctgcttccctgcagaactgcgctccaatctgcaaggaagccttctccctcaggacctcctcagccctgcctgccacatcccggccaccaggaggggttctcaggtactgggctcactctgtccctggaaaatcaacctctacagtctgggttagcttttcccttggtcttccagaaggtaattaattaggcaagtacatttttaaagaatttcaatctctctctctctctctctctctctctctctctccctctctctctctctctctctctctctctctctgtgtgtgtgtgtgtgtgtgagagagaaagagtatgtgagtccttaccttccacgtgttttgagaaacagtctcactgtgtacatcagagcttctggggactcacctgtctctgtctcattgtaggagggttggggtctcacacactctaccacatgcgtcctccctagtcccacttgccaatctggccctgtccctcctgctgtggggattctagggacacggaacctctactttctgccctcaggtggcccgccccttccctcctttcctgctctgaagactggggcaggcaggaaggaagcccgtagcctgggctgagcttgctcccccccccccgcccccctcccttgtgctaagcgctttcacttcctctgcttgttgcccgctgcaggagcatctcggtgcctgcgcccccgaccctccccatggctgacacttacgctgtggtgcagaagcgtggcgcttcggcgggcacagggccggggccgcgggcgcccaccagcacggacaccccgatctacagccaggtggctccacgtgcccagcgaccggtggcacacacggaggacgcacaggggacaacggcactgcgccgaggtgagttgaggccctccgggggtgtggagctgaggacaagtacctattagcaactttcccgctctcctaaggctcccgagggtctaagcgctgctcggaggtcagccccagatccagcatggccacgcccctaggctactaaacgtcggggttcaagccccaaaatgagccccaggtcaggtctcaattgggagactgggctgtctaacccaaggccccaaagacagacctccgaacttggacacgcccatggacacgcctcctaccgctacctcgggaagg^GAAATCGGTAGCTATGCTGCTCTAGAGTCCCTGGAACTCTTAAAAGCAGCTTGAGCTGACCCATAGGGGTTCTCCTGCCCTCAGTTCCTGCGGACCAAAACTCTTCCGGGCCTGATGCCTACGAAGAAGTAACAGATGGAGCACAGACTGGAGGGCTAGGTAAGTCAGAGCCTAGGGTGCAGCTTTGGAGAGCCCCAGGACTTCGGGAGTTCGGGCTGGGGC

This mutation is a 1972 bp deletion beginning at Chromosome 1 position 34,471,279 bp and ending after 34,473,250 bp (GRCm38/mm10). In addition, there is a 12 bp (GCACCCATTGCA) insertion at the deletion site

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
52772 TAA CCC AAG GCC CCA AAG A Wild type Forward A
52773 TGC TTT TAA GAG TTC CAG GGA C Common A
52774 CAC ACA AAA TGG GTG CAT ACT Mutant Forward A
52775 Fluorophore-1 AGA CCT CCG AAC TTG GAC ACG Quencher-1 WT Probe
52777 Fluorophore-2 CTG CAA TTG CAC CCA TTG C Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
52772 0.40 uM
52773 0.40 uM
52774 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.