Sep 28, 2023

Public workspaceDNA Cloning (Gibson Assembly, Transformation, Plating and Incubation) V.2

DOI
dx.doi.org/10.17504/protocols.io.8epv5xnddg1b/v2
  • 1National University of Singapore
NUS iGEM
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DOI: dx.doi.org/10.17504/protocols.io.8epv5xnddg1b/v2
Protocol Citation: NUS iGEM 2023. DNA Cloning (Gibson Assembly, Transformation, Plating and Incubation). protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5xnddg1b/v2Version created by NUS iGEM
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 28, 2023
Last Modified: September 28, 2023
Protocol Integer ID: 88545
Keywords: DNA Cloning, Gibson Assembly, Assembly, Plasmid Assembly, Transformation, Plating, E coli.
Abstract
2023 NUS-Singapore iGEM Team followed this protocol to construct the plasmid of interest or to clone the plasmid of interest.
Materials
  1. NEBuilder HiFi DNA Assembly Master Mix
  2. Competent cells made by 10β E. coli.
  3. LB media.

Safety warnings
  • Proper laboratory PPE must be worn at all times.
  • Thermal gloves shall be worn when handling competent cells from the -80°C fridge.
  • Since cells like 10β E. coli are used in this protocol, a Biosafety Cabinet (BSC) is required to ensure safety.
Gibson Assembly
Gibson Assembly
Calculate the volumes of respective DNA fragments to assemble based on their length and concentration. (The maximum final volume of the mixed fragments is Amount5 µL for each reaction.)

Add the DNA fragments into a PCR tube according to the volumes obtained from the calculation.

Add Amount5 µL of NEBuilder HiFi DNA Assembly Master Mix into the same PCR tube.

Vortex to mix the solution and centrifuge the PCR tube to spin down the remaining solution on the wall.
Incubate the PCR tube at 50°C for Duration01:00:00 .

1h
Transformation
Transformation
Switch on the water bath and set the temperature to 42°C.
Prepare a box of ice.
Take an Eppendorf tube that contains pre-made competent cells from the -80°C fridge.

Immediately place the Eppendorf tube with competent cells into the ice box for Duration00:05:00 .

5m
Add Amount20 µL of the Gibson Assembly product or Amount1 µL of pure DNA plasmid into the Eppendorf tube containing the competent cells.

Tap the bottom of the Eppendorf tube to mix the solution.
Leave the Eppendorf tube in ice for Duration00:10:00 .

10m
Place the Eppendoft tube into a foam floating.
Place them into the water bath for Duration00:00:45 at 42°C for heat shock.

45s
Place the Eppendorf tube into the ice immediately.
Add Amount1 mL of the LB media into the Eppendorf tube.

Place the Eppendoft tube into the incubator at 37°C for Duration01:00:00 for recovery.

1h
Centrifuge the Eppendorf tube to form a cell pellet (no specific speed and time).
Plating and Incubation
Plating and Incubation
1h
1h
Prepare an LB agar plate with the correct antibiotics.
Remove 950μL of the LB solution from the Eppendorf tube that contains the cell pellet, leaving about 100μL in the Eppendorf tube.
Resuspend the cells by pipetting the solution.
Spread the cells onto the agar with the L-spreader.
Place the petri dish in the incubator at 37°C for DurationOvernight to allow the colonies to grow.

1h