May 29, 2025
Molecular Cloning
- 1Icahn School of Medicine at Mount Sinai
Protocol Citation: Camille Goldman 2025. Molecular Cloning. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgqn1r3gk5/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: August 28, 2024
Last Modified: May 29, 2025
Protocol Integer ID: 106566
Keywords: ASAPCRN
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: ASAP-024297
NASA
Grant ID: 80ARC022CA004
NIH/NINDS
Grant ID: R01NS114239
NIH/NINDS
Grant ID: UH3NS115064
NIH/NIA
Grant ID: T32AG04968
NIH/NINDS
Grant ID: F31NS13090
Abstract
Protocol for cloning GEM-SCOPe plasmids via Gibson reaction
Design Cloning Reaction
Design Cloning Reaction
Acquire sequences for the plasmid backbone and inserts
Upload into NEBuilder Assembly Tool (nebuilder.neb.com) and follow the steps to get primers with 5' and 3' ends compatible for Gibson assembly
Order primers and necessary templates
Linearize Plasmid
Linearize Plasmid
Mix 5ug of backbone plasmid (pFUW; Addgene #14882) with 2 uL NheI-HF (NEB R3131), 2 uL BamHI-HF (NEB R3136), 5 uL 10x CutSmart Buffer.
Add nuclease free water to a final volume of 50uL
Incubate at 37°C for at least 1 hour
Heat inactivate at 65°C for 20 minutes
20m
Run the whole sample on a 1% agarose gel
Purify the desired gel fragment with a gel purification kit of choice
PCR Amplification of Inserts
PCR Amplification of Inserts
Combine 1 uL 10uM Forward Primer, 1 uL 10uM Reverse Primer, and 10 uL Q5 High-Fidelity 2X Master Mix (NEB M0493)
Add 20ng of template DNA
Add nuclease free water to a final volume of 20 uL
Repeat steps 7-9 for each insert
Run the PCR in a thermocycler. The following is for inserts of about 1000bp. Adjust annealing temperature and elongation time accordingly.
1. 98°C for 30s
2. Repeat the following 12x
- 98°C for 10s
- 72°C for 20s
- 72°C for 30s
3. Repeat the following 28x
- 98°C for 15s
- 60°C for 15s
- 72°C for 30s
4. 72° for 2m
5. Hold at 4°C
Run the PCR reactions on a 2% agarose gel to verify band size. Purify the gel fragments with a gel purification kit.
Gibson Assembly
Gibson Assembly
1h
1h
Add nuclease free water to a final volume of 10 uL
Combine 5uL HiFi DNA Assembly Master Mix (NEB E2621), 50 ng of linearized backbone with 3-fold molar excess of each insert (NEBioCalculator)
Prepare a negative control with 5uL HiFi DNA Assembly Master Mix, 50 ng of linearized backbone, and nuclease free water to a final volume of 10 uL.
Incubate at 50°C for 1h
1h
Purify Plasmid
Purify Plasmid
30m
30m
Thaw a tube of 10-beta Competent E. coli (NEB C3019) on ice for 10 minutes. Transfer 10uL into a fresh tube on ice.
Add 2uL of Gibson reaction to the bacteria and flick to mix
Keep the mixture on ice for 30m
30m
Heat shock at 42°C for 30s
30s
Place on ice for 5m
5m
Add 250uL of room temperature NEB 10-beta/Stable Outgrowth Medium
Place at 37°C for 60 minutes with vigorous shaking
1h
Meanwhile, warm up LB Agar plates with 100ug/mL ampicillin to 37°C
Spread 150uL of bacterial mixture onto plates
Incubate overnight at 37°C
Pick 1-3 colonies for mini prep isolation