Oct 09, 2023

Public workspaceGolden Gate Assembly V.2

DOI
dx.doi.org/10.17504/protocols.io.kqdg3xknqg25/v2
  • 1National University of Singapore
NUS iGEM
Open access
DOI: dx.doi.org/10.17504/protocols.io.kqdg3xknqg25/v2
Protocol Citation: NUS iGEM 2023. Golden Gate Assembly. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg3xknqg25/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: October 08, 2023
Last Modified: October 09, 2023
Protocol Integer ID: 88975
Keywords: Golden Gate, Assembly, DNA Assembly, DNA, BsaI, Restriction site, Restriction enzyme
Abstract
2023 NUS-Singapore iGEM team followed this protocol to assemble DNA oligos containing Golden Gate restriction sites with a plasmid backbone that contains the same restriction sites. The restriction enzymes utilised in this protocol are the Type IIS restriction enzymes, specifically BsaI. The use of Type IIS restriction enzymes ensures that there is no scar or extra sequence at the junctions between the assembled fragments.
Guidelines
This protocol outlines the Golden Gate procedures with a sample volume of 20 µL per reaction.
Protocol materials
ReagentBsaI-HF®v2New England BiolabsCatalog #R3733S
Step 2
ReagentT4 DNA LigaseNew England BiolabsCatalog #M0202S
Step 2
Reagent10X NEB T4 DNA ligase bufferNew England Biolabs
Step 2
Safety warnings
Attention
  • Proper lab PPE must be worn at all times.
  • Thermal gloves shall be worn when handling items from Temperature-20 °C fridge.

Golden Gate Assembly
Golden Gate Assembly
Prepare an ice box.
Place the Reagent10X NEB T4 DNA ligase bufferNew England Biolabs , ReagentBsaI-HF®v2New England BiolabsCatalog #R3733S , and ReagentT4 DNA LigaseNew England BiolabsCatalog #M0202S in ice.

Add the following reagents into a PCR tube:

ItemVolume
DI Water10μL
Plasmid1μL
PCR Extract Oligo5μL
BsaI-HFv2 Enzyme1μL
T4 DNA Ligase1μL
T4 Ligase Buffer2μL

Note
Reagents with enzymes such as BsaI-HFv2 Enzyme and T4 DNA Ligase must be kept at a low temperature (in ice) when they are in-use to prevent the enzymes from denaturation.

Put the sample into the Thermal Cycler and run it with the following conditions:

*Set "Lid Temperature" to Temperature105 °C and set "Volume" to Amount20 µL
TemperatureDuration
37°C5 minutes
16°C5 minutes
Go to step 1, repeat the cycle 40 times
37°C1 hour
60°C15 minutes
12°CInfinite Loop

Transformation
Transformation
1h 15m 45s
Prepare a box of ice.
Take an Eppendorf tube that contains pre-made competent cells from the Temperature-80 °C fridge.

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

5m
Add the whole Golden Gate Assembly product (Amount20 µL ) o 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 Temperature42 °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 Temperature37 °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
Prepare an LB agar plate with the correct antibiotics.
Remove Amount950 µL of the LB solution from the Eppendorf tube that contains the cell pellet, leaving about Amount100 µL 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 Temperature37 °C for DurationOvernight to allow the colonies to grow.

1h