Question

Asked 10th Jul, 2021

Efficiency of Gibson cloning vs Golden Gate?

I have been using NEB Hifi Gibson assembly for a couple years now and I've been quite happy with it. I regularly make plasmid constructs with 4-8 fragments, and always >1/4 of the colonies are "perfect," while the remaining ones may have some SNPs at the joining sites, or be misassembled due to a repetitive region.

Some colleagues said that Golden Gate has even higher efficiency. That even with 8 fragments, it is normal for 50-100% of colonies to be perfect. Is that true? Is Golden Gate that perfect?

Sequence and Ligation Independent (SLIC) Cloning

Molecular Cloning

Bandla Ramesh

Centurion University of Technology and Management

The efficiency of Gibson cloning is higher than that of Golden Gate. Gibson cloning is a one-step assembly method that uses a DNA ligase enzyme to join two or more DNA fragments together. It is highly efficient, with reported success rates of up to 95%. Golden Gate cloning is a two-step assembly method that relies on a restriction enzyme and a DNA ligase to join two or more DNA fragments together. This method is less efficient, with reported success rates of up to 80%.

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Simon Miguel Lopez

Academia Sinica

Hello Gen

Based on my understanding, I believe that it really depends on the kind of experiment that you are doing. Gibson method is considered as a "gold-standard" for assembly of DNA fragments, and has certain advantages over Golden-Gate method and vise versa. Based on the papers and discussions that I had with my colleagues, we find these kinds of questions challenging since we do not absolutely know which is the best method for DNA assembly. Each of them has its own advantages and disadvantages, and may be appropriate on a particular type of study. While most people recognize that Gibson has the gold-standard for assembly, other methods can be explored by conducting experiments.

Hope this helps.

Best.

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Avinash Marwal

Mohan Lal Sukhadia University

https://bitesizebio.com/26961/cloning-methods-5-different-ways-to-assemble/

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Ladan Mashouri

University of Texas at Austin

Hi everyone,

I am just trying to do it with 4 inserts and a double digested vector with BsaI and BbsI. The information about vectors is as below:

1. 208 bps (17 bp overlap with vector)

2. 1254 bps (20 bp overlap with 1)

3. 815 bps (20 bp overlap with 2)

4. 114 bps (25 bp overlap with vector)

I used different molar ratios using NEB calculator:

I tried 1:2 for all

1:1 for fragment longer than 1000bps

1:5 for lower than 200 bps

and different combination of all

1 hour of incubation at 50 celsius. And also used both 10 µl and 20 µl reaction and finally transformed 5 µl of it to the competent cells.

but no colonies...

Would you please help me with it?

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Bandla Ramesh

Centurion University of Technology and Management

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Asked 24th Nov, 2022

Florian Pruckner

Hi,

I am having troubles cloning a Cas9 gene (around 4300 kb) into a vector, replacing a LacZ cassette. (I use the uloop system.) I use SapI as a restriction enzyme and T4 as a ligase. I cloned several other constructs into the same kind of vector with high efficiency (mainly white colonies, and almost all carry the correct insert when screened).

When I try to insert Cas9 (or also dCas9) I yield the same number of colonies, with again almost all white. However, when I screen the colonies via colony PCR or restriction digest the isolated plasmids of the colonies, no vector carries the insert. I did not sequence these plasmids, but from the restriction digest it suggest, that the plasmid only lost its LacZ cassette and was closed again without any insert (eventhough overhangs are not compatible).

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37 C 5min...............37 C 5min

16 C 5min................16 C 5min

(25cycles)................(40cycles)

65 20min-................65 20min

85 10min..................85 10min

4 hold......................4 hold

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-T4 buffer only

-Or 50% T4 buffer, 50% Cutsmart buffer

I started with fresh Cas9 (and dCas9) PCR templates and with several fresh receiver plasmids. Also I used different competent cell batches.

I double checked the overhangs of receiver plasmids and the overhangs created on the Cas9 insert.

I am running out of ideas..

Does anyone know if GGC with single large inserts (4000 kb+) effect GGC efficiency?

Does anyone have a suggestion how I could improve GGC efficiency?

Further troubleshooting suggestions?

I really would appreciate your ideas!

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Florian

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The next sensible thing would be that my ligation is simply very difficult for some reason, but in that case, it wouldn't be failing for all 4 constructs.

I linearise my vectors by PCR and gel-purify them. I have tried with purified and unpurified PCR products.

I tried assembling the positive control in the HiFi mix and got 3-5 colonies, which is still way too little (manual says >100). So I used another vial of HiFi mix, thinking maybe the enzymes were bad, but I got exactly the same result.

I've used this same method multiple times in the past to assemble constructs of higher complexity than this, and it has always worked in the past for me... What am I missing here? Should I sacrifice a loved one?

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