Sep 30, 2023

Public workspacePlasmid Construction Protocol

This protocol is a draft, published without a DOI.
  • 1University of California, Berkeley;
  • 2Aligning Science Across Parkinson's
Annan SI Cook
Open access
Protocol Citation: Xuefeng Ren, Annan SI Cook 2023. Plasmid Construction Protocol. protocols.io https://protocols.io/view/plasmid-construction-protocol-c2n7ydhn
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: September 25, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 88511
Keywords: ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson's
Grant ID: ASAP-000350
Abstract
This protocol details plasmid construction in general terms for the insertion of genes into the pCAG mammalian expression vector.
Attachments
852-2204.pdf
852-2204.pdf
31KB
Materials
Materials

  • DNA sequences encoding PI3KC3-C1 subunits and VPS15 mutants (codon-optimized and synthesized)
  • pCAG vector
  • Restriction enzymes (e.g., New England Biolabs enzymes)
  • Gibson assembly kit
  • Sanger sequencing service
Design Plasmid Constructs
Design Plasmid Constructs
This protocol uses the pCAG vector and NEB restriction enzymes and Gibson Assembly kits.
Obtain the DNA sequences for the genes of interest from your preferred DNA synthesis vendor.
Digestion or Gibson Assembly
Digestion or Gibson Assembly
Decide whether to use restriction digestion or Gibson assembly for subcloning.
Restriction Digestion:
Digest both the pCAG vector and the DNA fragments (genes/tags) using appropriate restriction enzymes.
Digestion
Purify the digested fragments using a DNA purification kit.
Gibson Assembly:
Follow the manufacturer's protocol for the Gibson assembly kit to assemble the DNA fragments into the pCAG vector.
Ligation
Ligation
If using restriction digestion, perform a ligation reaction to insert the digested DNA fragments into the linearized pCAG vector.
Use a DNA ligation kit and follow the manufacturer's instructions.
Transformation
Transformation
Transform the ligated plasmids into competent E. coli cells.
Plate the transformed cells on selective agar plates containing the appropriate antibiotic for plasmid selection.
Bacterial Culture
Bacterial Culture
Incubate the plates DurationOvernight at an appropriate temperature for E. coli growth (e.g., Temperature37 °C ).

Incubation
Overnight
Plasmid Isolation
Plasmid Isolation
Pick colonies that have grown on the plates and inoculate them into liquid culture with the same antibiotic.
Grow the cultures to obtain a sufficient amount of plasmid DNA.
Isolate the plasmid DNA using a plasmid purification kit.
Sanger sequence the results.