Cloning and Mutagenesis of TPP1

The TPP1 coding sequence was previously cloned into a pmCherry-N1 vector
backbone (Clontech, CA) in place of the pmCherry coding
sequence (23 (link)). Desired changes were
introduced utilizing “round the horn site-directed mutagenesis”
(24 (link)) with a DpnI restriction digest
included to remove parental template. Primers were purchased from Integrated DNA
Technologies Inc. and molecular biology enzymes were purchased from New England
Biolabs. Mutations were verified by sequencing (Macrogen) and DNA for
transfection was purified using the PureYield Plasmid MaxiPrep System
(Promega).

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Collier A.M., Nemtsova Y., Kuber N., Banach-Petrosky W., Modak A., Sleat D.E., Nanda V, & Lobel P. (2020). Lysosomal protein thermal stability does not correlate with cellular half-life: Global observations and a case study of tripeptidyl-peptidase 1. The Biochemical journal, 477(3), 727-745.

Publication 2020

PureYield Plasmid MaxiPrep System

Coding sequence Enzymes Horn Mutations Parental Plasmid Primers Promega Site directed mutagenesis

Corresponding Organization : Johnson University

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Variable analysis

independent variables

Introduced desired changes utilizing 'round the horn site-directed mutagenesis'

dependent variables

Mutations were verified by sequencing

control variables

TPP1 coding sequence was previously cloned into a pmCherry-N1 vector backbone (Clontech, CA) in place of the pmCherry coding sequence
DpnI restriction digest was included to remove parental template
Molecular biology enzymes were purchased from New England Biolabs
DNA for transfection was purified using the PureYield Plasmid MaxiPrep System (Promega)

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