Golden Gate Assembly (Esp31 or Bsa1 v2 HF)
Jamie Auxillos, Edward Wallace
Abstract
This protocol is for the assembly of DNA parts using Golden Gate assembly (Engler et al. 2008) with modifications from on publication by Garcia-Ruiz et al. (2018) and online resource by NEB (https://international.neb.com/protocols/2018/10/02/golden-gate-assembly-protocol-for-using-neb-golden-gate-assembly-mix-e1601).
Parts can either be assembled by Golden Gate assembly using Esp31 or Bsa1v2-HF depending on the restriction sites flanking the parts. Using this protocol, multiple insert parts can be assembled into the parts acceptor vector. The Golden Gate assembly reaction is incubated in a thermocycling program set at a digestion temperature of at 37℃ and ligation temperature of 16℃ for 20-30 cycles. The reaction is subsequently transformed into E. coli cells (DH5ɑ, Mach1, TOP10, NEB10β).
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Engler C, Kandzia R, Marillonnet S (2008)A One Pot, One Step, Precision Cloning Method with High Throughput Capability. PLOS ONE 3(11): e3647. doi: 10.1371/journal.pone.0003647
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Garcia-Ruiz E, Auxillos J, Li T, Dai J, Cai Y. YeastFab: High-Throughput Genetic Parts Construction, Measurement, and Pathway Engineering in Yeast. Methods Enzymol. 2018;608:277-306. doi: 10.1016/bs.mie.2018.05.003.
Steps
Measure the concentration of your insert and vector DNA using a microvolume spectrophotometer (e.g. nanodrop).
Calculate the volumes required to mix equimolar quantities of these. Multiple insert parts can be assembled into the parts acceptor vector.
Note - This master mix needs to be prepared on ice and kept on ice!
Taking into consideration the volume for the insert and vector DNA, prepare the following Golden Gate master mix for a final reaction volume of 10µL. Prepare the master mix according to the table below in a 1.5 ml eppendorf tube.
- Scale up the mix below based on the number of assemblies you would like to set up.
- e.g. if the total volume of insert and vector DNA is 3 µl, prepare a master mix with a volume of 5.45 µl of water.
| A | B |
|---|---|
| Reagents | For 1 reaction (μl) |
| 10x T4 Buffer | 1 |
| T4 DNA Ligase (2,000,000 U/mL) | 0.05 |
| Esp31 or Bsa1v2-HF | 0.5 |
| Water | To a total volume of 10 µl |
Label PCR tubes (both on the side and on the lid) then place the tube on a PCR tube rack on ice.
Note - This step needs to be carried out on ice!
Aliquot the Golden Gate assembly master mix into each well on a PCR tube strip
Note - This step needs to be carried out on ice!
Add the appropriate volume of vector DNA to the master mix, as calculated in step 1.
Note - This step needs to be carried out on ice!
Add the appropriate volume of insert DNA to the master mix, as calculated in step 1. The total reaction volume should be 10µL.
Mix the reaction by flicking the tubes and briefly spin on a PCR tube spinner. Transfer tubes back on ice.
Set up the following Golden Gate assembly program on a thermocycler:
| A | B | C | D |
|---|---|---|---|
| Step 1 | 37℃ | 5 minutes | |
| Step 2 | 37℃ | 5 minutes | 30x |
| Step 3 | 16℃ | 10 minutes | |
| Step 4 | 16℃ | 30 minutes | |
| Step 5 | 37℃ | 60 minutes | |
| Step 6 | 50℃ | 5 minutes | |
| Step 7 | 80℃ | 10 minutes | |
| Step 8 | 4℃ | ∞ |
Transfer PCR tubes to the thermocycler and start the program.
An optional step if your end-resulting plasmid does not have internal BsaI/Esp31 sites within the insert or backbone vector (other than the ones used for the assembly of insert and vector), you can add ATP-dependent DNase (Lucigen E3101K - Plasmid-Safe™ ATP-Dependent DNase) which digests linear DNA but not circular DNA. Add 0.25µL of Plasmidsafe DNase and 0.5µL of 25 mM ATP to the Golden Gate reaction and incubate at 37°C for 0h 15m 0s
Transform the Golden Gate reaction into E. coli cells (e.g. DH5ɑ, Mach1, TOP10, NEB10β), following an appropriate protocol.
