Dec 09, 2024

Public workspaceDesigning Overexpression Plasmids for Cellular Studies

DOI
https://dx.doi.org/10.17504/protocols.io.36wgqd3y3vk5/v1
  • Mukesh Kumar1,
  • Timothy A. Ryan1,2
  • 1Department of Biochemistry, Weill Cornell Medicine, New York, NY 10065;
  • 2Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, Maryland 20815, USA
Mukesh Kumar
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DOI: https://dx.doi.org/10.17504/protocols.io.36wgqd3y3vk5/v1
Protocol Citation: Mukesh Kumar, Timothy A. Ryan 2024. Designing Overexpression Plasmids for Cellular Studies. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgqd3y3vk5/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: November 26, 2024
Last Modified: December 09, 2024
Protocol Integer ID: 114390
Keywords: ASAPCRN, designing overexpression plasmids for cellular study, designing overexpression plasmid, function in primary cultured neuron, primary cultured neuron, cellular study, protein localization, studies of protein localization, using gibson assembly, neuron, gibson assembly
Funders Acknowledgements:
NIH
Grant ID: NS036942
NIH
Grant ID: NS11739
ASAP
Grant ID: ASAP-024404
Abstract
This protocol provides a detailed stepwise method for generating fluorescently tagged constructs using Gibson Assembly, enabling studies of protein localization and function in primary cultured neurons.
Materials
Plasmids and Genes:

  • ReagentCMV mRuby3-Synapsin1aaddgeneCatalog #187896
  • ReagentDDHD2GenscriptCatalog #OHu23306
  • ReagentCPT2GenscriptCatalog #OHu17939D

Vectors and Promoters:

  • Reagentplenti hSyn PGK1-HALOaddgeneCatalog #220910

  • Restriction Enzymes: AgeI, SalI, XhoI, PmeI (Acquired from NEB)

  • ReagentQIAquick Gel Extraction Kit (250)QiagenCatalog #28706

  • Gibson Assembly: ReagentNEBuilder HiFi DNA Assembly Master Mix - 50 rxnsNew England BiolabsCatalog #E2621L

  • PCR Reagents: High-fidelity DNA polymerase

NEB Stable Competent E. coli:

  • ReagentNEB Stable Competent E.coli (High Efficiency) - 20x0.05 mlNew England BiolabsCatalog #C3040H

  • PCR machine
  • Gel electrophoresis apparatus
  • UV gel imager
  • Heat block or thermal cycler

Troubleshooting
PCR Amplification of Inserts
Design primers for PCR amplification of target genes, ensuring the inclusion of overlapping sequences compatible with Gibson Assembly and restriction sites as needed:
eGFP-DDHD2: From DDHD2_OHu23306C_pcDNA3.1(+)-N-eGFP, add XhoI and PmeI.
CPT2: From CPT2_OHu17939D_pcDNA3.1+/C-(k)DYK, add XhoI and PmeI.
Amplify target genes using a high-fidelity DNA polymerase to minimize errors.
Verify the PCR product sizes via gel electrophoresis and purify the bands using gel extraction and cleanup kit.
Restriction Digestion of Vectors
Digest the pLV vector with appropriate restriction enzymes:
For mRuby-Synapsin: AgeI and SalI.
For eGFP-DDHD2 and CPT2: XhoI and PmeI.
Resolve the digested vector on an agarose gel and purify the linearized vector using Qiagen gel extraction kit.
Gibson Assembly Reaction
1h
Prepare the Gibson Assembly reaction using NEBuilder® HiFi DNA Assembly Master Mix:
Mix linearized pLV vector and purified PCR-amplified inserts at a 1:3 molar ratio.
Mix
Add Amount10 µL of NEBuilder® HiFi DNA Assembly Master Mix.

Pipetting
Adjust the total reaction volume to Amount20 µL with nuclease-free water.

Incubate the reaction at Temperature50 °C for Duration01:00:00 .

1h
Incubation
Temperature
Transformation of Competent E. coli
1h
Transform Amount5 µL -Amount10 µL of the Gibson Assembly reaction into competent E. coli.

Plate the transformed cells on LB agar plates with appropriate antibiotics.
Incubate the plates DurationOvernight at Temperature37 °C .

1h
Incubation
Overnight
Temperature
Verification of Clones
Screen colonies by colony PCR or restriction digestion to confirm successful assembly of the constructs.
Sequence positive clones to verify the integrity of the constructs.
Plasmid Preparation
1h
Grow positive colonies in LB broth with antibiotics DurationOvernight .

1h
Extract plasmid DNA using a plasmid purification kit.

Note
• Primer design is critical for successful Gibson Assembly; ensure adequate overlapping sequences (>15 bases) for seamless assembly.
• Use high-fidelity enzymes for PCR to prevent mutations in amplified inserts.
• Handle restriction enzymes and Gibson Assembly reagents on ice to maintain activity.
• Verify plasmid sequences to ensure correct insert orientation and integrity.