Oct 22, 2021
in vitro assembly and transformation
This protocol is a draft, published without a DOI.
- Yuichiroh Ikagawa1
- 1iGEM Gifu
- iGEM Gifu
Protocol Citation: Yuichiroh Ikagawa 2021. in vitro assembly and transformation. protocols.io https://protocols.io/view/in-vitro-assembly-and-transformation-bzc9p2z6
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: In development
We are still developing and optimizing this protocol
Created: October 21, 2021
Last Modified: October 22, 2021
Protocol Integer ID: 54401
Keywords: dna assembly, assembly of the dna fragment, dna ligase, cloning, dna fragment, cloning efficiency, ends of the dna fragment, dna polymerase, cyclized plasmid, high molecular weight plasmid, dna, gibson assembly, advantage over other strain, assembly kit, ligase, assembly, strain, other strain, cell, competent cell, plasmid
Abstract
For the in vitro assembly of the DNA fragments, we decided to use the NEBuilder®, an assembly kit based on the Gibson Assembly. In NEBuilder®, exonucleases break down the ends of the DNA fragments and hybridize the cohesive ends, followed by DNA polymerases that synthesize the broken strands. Finally, DNA ligase repairs the nick and completes the DNA assembly. it is expected that in vitro assembly will reliably transform the cyclized plasmid.
For the subsequent transformation, we used the NEB® 10-beta competent cell because NEB® 10-beta can be largely maintained the cloning efficiency even with high molecular weight plasmids, which we considered to be an advantage over other strains when transforming our team's plasmids.
Materials
Reagents
DNA samples
- Cas12a fragment 1
- Cas12a fragment 2
- Cas12a fragment 3
- pSB1A3
NEBuilder® Assembly Master Mix (New England Biolabs)
NEB® 10-beta competent cell (New England Biolabs)
SOC medium
LB agar plate
in vitro assembly
Thaw the DNA solution and NEBuilder® Assembly Master Mix on ice.
Mix the DNA solution by vortexing, and centrifuge to collect the solution to the bottom of the tube.
DNA solutions and reagents were mixed according to the compositions in the table below.
| A | B | C | |
| COMPONENT | VOLUME(µl) | CONCENTRATION | |
| NEBuilder Assembly Master Mix | 10.0 | ×2 | |
| Cas12a fragment 1 | 0.75 | 0.34 pmol | |
| Cas12a fragment 2 | 0.90 | 0.35 pmol | |
| Cas12a fragment 3 | 1.00 | 0.33 pmol | |
| pSB1A3 | 0.60 | 0.36 pmol | |
| Nuclease Free water | 6.75 | ||
| Total Volume | 20.0 |
Incubate at 37℃ for 1 hour.
transformation
Thaw Escherichia coli NEB® 10-beta competent cell on ice.
Add 2 μl assembled sample into competent cell tube.
Mix gently pipetting 4~5 times.
Incubate on ice for 30 minutes.
Heat shock at 42℃ for 30 seconds on heat block.
Incubate on ice for 2 minutes.
Add 950 μl of SOC medium and mix gently pipetting.
Incubate the tube at 37℃ for 1 hour.
Centrifuge at 5,000 g for 1 minute at room temperature
Discard 900 μl of the supernatant.
Resuspend cells with the remaining supernatant by Voltex
Spread the whole culture on agar plates containing ampicillin.
Incubate overnight at 37℃