Jan 30, 2023
Golden Gate Assembly
This protocol is a draft, published without a DOI.
- 1San Diego State University
Protocol Citation: Amanda T Alker, Nicholas J Shikuma 2023. Golden Gate Assembly. protocols.io https://protocols.io/view/golden-gate-assembly-b5i2q4ge
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: February 23, 2022
Last Modified: January 30, 2023
Protocol Integer ID: 58682
Keywords: Cloning, Golden Gate Assembly, Plasmid
Funders Acknowledgement:
Gordon and Betty Moore Foundation
Grant ID: GBMF9344
National Science Foundation
Grant ID: 1942251
Office of Naval Research
Grant ID: N00014-20-1-2120
Abstract
Protocol for golden gate assembly of modular plasmids from Yeast Toolkit, Bee Toolkit and Marine Modification Kit platforms.
Day 1
Day 1
Streak out the plasmid parts that will be used in the assembly onto LB agar plates with the appropriate antibiotics (Chloramphenicol 100µg/mL for Type 1-7 parts; Kanamycin or Gentamicin 100µg/mL for Type 8 backbone parts). Incubate overnight at 37ºC.
Day 2
Day 2
Inoculate a single colony into 25mL of LB plus antibiotics in the late afternoon/early evening. Incubate overnight at 37ºC while shaking at 200rpm.
Day 3
Day 3
Spin the culture at 5000g for 20 minutes. Remove the supernatant, resuspend the pellet in 1mL water. Perform a plasmid miniprep (Zyppy miniprep or Omega E.Z.N.A. Plasmid Mini Kit II) following the standard kit protocols.
Measure the Plasmid DNA concentration on a spectrophotometer.
Perform the Golden Gate Assembly:
Dilute backbone plasmid parts and add 10fmol to the reaction
Dilute the insert plasmid parts and add to 20fmol of each insert to the reaction
Add 2µL T4 Ligase Buffer (Promega)
Add 1µL T4 Ligase
Add 1µL BsaI-HF_V2 or BsmBI-HF endonuclease
Add X water up to a 20µL reaction
Note
Follow the reaction protocol listed on the Barrick Lab website:
Run the thermocycler program for BsaI/BsmBI as follows:
| A | B | C | |
| Step | Temperature | Time | |
| 1 | 37/42ºC | 5 minutes | |
| 2 | 16ºC | 5 minutes | |
| Cycles 1-2 | Repeat 30x | ||
| 3 | 37/55ºC | 10 minutes | |
| 4 | 80ºC | 10 minutes |
Note
The thermalcycler program used for this protocol can be found on the Barrick Lab website here, along with other great troubleshooting tips.
Day 4
Day 4
Dilute 2x or electroporate 2µL of the GGA directly into electrocompetent cells (i.e. SM10pir, S17pir). Recover 1+ hours and plate on LB Agar media containing the correct antibiotic concentrations. Incubate at 37ºC overnight.
Day 5
Day 5
Screen colonies for correct insert and perform colony PCR with primers spanning assembly junctions.
Streak out clones that yield a band onto LB Agar media containing the correct antibiotic concentrations and incubate at 37ºC overnight.
Day 6
Day 6
Inoculate a single colony into 25mL LB broth containing the correct antibiotic concentrations and incubate at 37ºC overnight.
Day 7
Day 7
Store overnight culture in cryovials for long term storage. Add 500µL of culture to 500µL of 50% glycerol and store in -80ºC freezer.
With remaining culture, Centrifuge at 5000g for 20 minutes. Remove the supernatant, resuspend the pellet in 1mL water. Perform a plasmid miniprep (Zyppy miniprep or Omega E.Z.N.A. Plasmid Mini Kit II) following the standard kit protocol. Elute with water.
Measure the Plasmid DNA concentration on a spectrophotometer.
Send the miniprepped plasmid for Sanger or Oxford Nanopore long-read plasmid sequencing to confirm the construct. Store the remaining miniprep for downstream applications (i.e. shuttle into different electrocompetent cell for conjugation).