Golden Gate Assembly
NUS iGEM
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.
Steps
Golden Gate Assembly
Prepare an ice box.
Place the
Add the following reagents into a PCR tube:
| A | B |
|---|---|
| DI Water | 10μL |
| Plasmid | 1μL |
| PCR Extract Oligo | 5μL |
| BsaI-HFv2 Enzyme | 1μL |
| T4 DNA Ligase | 1μL |
| T4 Ligase Buffer | 2μL |
Put the sample into the Thermal Cycler and run it with the following conditions:
*Set "Lid Temperature" to 105°C and set "Volume" to 20µL
| A | B |
|---|---|
| 37°C | 5 minutes |
| 16°C | 5 minutes |
| Go to step 1, repeat the cycle 40 times | |
| 37°C | 1 hour |
| 60°C | 15 minutes |
| 12°C | Infinite Loop |
Transformation
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 0h 5m 0s .
Add the whole Golden Gate Assembly product (20µL) 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 0h 10m 0s .
Place the Eppendoft tube into a foam floating.
Place them into the water bath for 0h 0m 45s at 42°C for heat shock.
Place the Eppendorf tube into the ice immediately
Add 1mL of the LB media into the Eppendorf tube.
Place the Eppendoft tube into the incubator at 37°C for 1h 0m 0s for recovery.
Centrifuge the Eppendorf tube to form a cell pellet (no specific speed and time).
Plating and Incubation
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 1h 0m 0s to allow the colonies to grow.
