Sep 23, 2023
Plasmid Construction and Gibson cloning
- 1Sascha Martens lab, University of Vienna, Max Perutz Labs - Vienna
Protocol Citation: Elias Adriaenssens 2023. Plasmid Construction and Gibson cloning. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5x11ng1b/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 28, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 84153
Keywords: Plasmid Construction, ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson’s (ASAP)
Grant ID: ASAP-000350
Marie Skłodowska-Curie MSCA Postdoctoral fellowship
Grant ID: 101062916
Abstract
This protocol describes plasmid construction.
Attachments
759-1927.pdf
43KB
Plasmid Construction
Plasmid Construction
5h
Obtain the sequences of all cDNAs by amplifying existing plasmids, HAP1 cDNA, or through gene synthesis (Genscript).
For insect cell expressions, the sequences are codon optimized and the gene is synthesized (Genscript).
With the exception of the NAP1-6xAla mutant, which was obtained through gene synthesis (Genscript), all other plasmids were generated by Gibson cloning.
For Gibson cloning, generate inserts and vector backbones by PCR amplification or excise from agarose gels after restriction enzyme digestion at 37 °C for 02:00:00 .
2h
Purify the inserts and plasmid backbones with Promega Wizard SV gel and PCR Cleanup System (Promega).
Mix the purified inserts and backbones in a molar 3:1 ratio, respectively, supplemented by a 2x NEBuilder HiFi DNA assembly enzyme mix (New England Biolabs).
Incubate Gibson reactions for 01:00:00 at 50 °C and then transform into DH5-alpha competent E. coli cells.
1h
Grow transformed Gibson reactions Overnight on agar plates containing the appropriate selection marker (ampicillin, kanamycin, or chloramphenicol).
1h
Pick single colonies, grown Overnight in liquid cultures, and pellet for DNA plasmid extraction using the GeneJet Plasmid Miniprep kit (Thermo Fisher).
1h
Submit the purified plasmid DNA for DNA Sanger sequencing (MicroSynth AG) to verify insert sequences by Sanger sequencing.
Further analyze positive clones by whole plasmid sequencing (Plasmidsaurus).