In vitro transcription of mRNAs

The mRNAs and 4.5S RNA were transcribed in vitro from linear DNA templates and purified by ion exchange chromatography27 (link). The DNA templates were PCR-amplified using the pUC19 plasmid (ThermoFisher Scientific) (for proOmpA, TolB, DsbA and 4.5S RNA) or the pET24a plasmid (Novagen) (for HemK, LepB and LepB variants) carrying the respective gene, using Phusion polymerase (NEB) together with a forward primer binding to the T7 promoter and a reverse primer placed according to the desired length of the mRNA6 (link)28 (link). The RNaseH construct was amplified from pET24a plasmid using analogous procedures.

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Ranjan A., Mercier E., Bhatt A, & Wintermeyer W. (2017). Signal recognition particle prevents N-terminal processing of bacterial membrane proteins. Nature Communications, 8, 15562.

Publication 2017

pUC19 plasmid Phusion polymerase

4 5s rna Gene Ion exchange Mrnas Plasmid Primer

Corresponding Organization :

Other organizations : Max Planck Institute for Biophysical Chemistry

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Variable analysis

independent variables

Linear DNA templates
Primers for PCR amplification

dependent variables

MRNAs
4.5S RNA

control variables

PUC19 plasmid
PET24a plasmid
Phusion polymerase

controls

Positive control: pUC19 plasmid and pET24a plasmid were used to generate the DNA templates.
Negative control: Not explicitly mentioned.

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