I used Gibson assemblies to create an AAV-compatible plasmid. Given the high recombination rate associated with constructs containing ITRs, I tried to transform my assembly product in One shot Stbl3 (transformation efficiency > 1x10^8) and consistently got no clones. I then tried high efficiency DH5alphas (transformation efficiency > 1x10^9) and I get a lot of clones.
I now transform my constructs in these DH5 alpha cells, expand minimally at low temperatures and miniprep. I then use the miniprep DNA to transform Stbl3 (which works beautifully) and expand with these stabler cells.
As this protocol works for me, I would like to know if other people have problems transforming Stbl3 with Gibson products. Their transformation efficiency is reportedly high, And I found them to be very efficient when I transform from restriction digestion/ligation products.
If useful, my Gibson assembly is made from 2 fragments (Vector + insert), I typically use ~50ng of vector and ~60ng insert (4x excess), and run the reaction for 1h.
Is it typical to use very efficient cells to transform from a Gibson?
Should I try running the reaction with more DNA?
Thanks!
We compared Stabl3 side by side with NEB Stable cells and difference in transformation efficiency was quite unexpected - about 2 orders of magnitude in favor of NEB cells.
I would avoid cells not specifically tweaked to handle large DNA or DNA containing iTRs just to be safer.
There is a caveat with NEB cells - they grow slower and form smaller colonies. This in itself is not a deal-breaker but if you are interested in making large quantity of DNA, your yield of cells from an overnight growth, and consequently of plasmid DNA, will be low. If necessary, once you have isolated and identified the plasmid(s) from NEB cells, you can always transfer them to Stabl3.
Enjoy feeding the two cookie monsters!
There is an incompatibility between the Gibson assembly buffer and that of the Stbl3 cells such that the Stbl3 cells are not able to take up your plasmid and therefore all die under antibiotic selection (if you talk to NEB they will tell you this). I have experienced this problem myself and use ethanol precipitation to clean up my assembly product before transforming into Stbl3 cells. It's been my experience that the NEB version of Stbl3 cells are not really that stable, so depending on the construct, cleaning up might be worth it. Hope this helps.
Hello everyone,
I posted this question earlier as well but have not got much response.
As you all have used Gibson assembly kit. Please guide me through this.
I am planning to perform Gibson assembly for splicing 3 fragments and finally want them circularised. Previously I was performing overlap extension PCR but that did not work for me, I guess the reason being the size of fragments(>4.0 kb each).
Previously I was performing overlap extension PCR but that did not work for me, I guess the reason being the size of fragments(>4.0 kb each).
I have designed primers for Gibson assembly using SnapGene. The primers are GC rich upto 60%. And I have also amplified the fragments successfully.
How should I go about this cloning procedure? Do you have any suggestion?
I am so behind on this but better late than never:
Tausif and Natalie, thank you very much for your helpful responses.
Natalie: I have cleaned my DNA and now it works, you shaved a few steps off my protocols, thanks!
Vedita: If I understand correctly, you manage to generate your fragments by PCR but either the assembly or the transformation does not work... is that correct? 3 fragments of 4-5 KB should not be a problem for Gibson assembly. How long are your overlaps? If you are dealing with a GC-rich region, you could consider shortening or elongating your overlapping sequence so that the 10 bp at the very end have a lower TM.
Also, which competent cells are you using? If you are already using high-efficiency cells, you could consider purifying and concentrating your assembly product.
Hope this helps!
Hello Simon,
I am using 30-35 bp overlap and the overlap region is of 60 to 65%GC content.
Gibson kit from NEB has competent cells.
So, my experimental plan is this: I have got 3 fragments 4.1 kb, 4.3 kb, 4.9 kb each. I have generated amplified products using PCR. I will purify each fragment. I will use Gibson assembly to assemble them together in a way that the finally there will be circular assembled product.
So, after assembly, how will I check that assembly has happened? And should I first assemble 2 fragments together and in other reaction, I should add the 3rd fragment?
Hi Vedita,
I would assemble the three fragments in a single reaction.
Make sure you have equal molar amounts of each (corrected for length) and let the reaction go for 60 minutes.
If that doesn't work, my first go to would be to clean and concentrate the DNA with a kit such as the Monarch DNA cleanup kit.
I would also verify the TM and self complementarity of the 10 last BP of your fragments. If it is too GC rich and you have self complementarity, you could "walk backwards" and prune the tips until the last 10 have a better profile. I have consistently assembled fragments with 15 BP overlap, so you have room to cut from your 30-35 design.
Hope this helps!
Thank you, Simon, for your suggestions.
I have amplified the fragments via PCR and I will also gel purify them.
Hey everyone,
I had set up 3 reactions, one for Gibson's assembly, HiFi assembly and a positive control(all from NEB). I had used 0.05 pM concentration for each fragment (4.1 kb each) and are gel purified. After the reaction for 1 hr at 50 0C, I did the transformation of NEB Chemically competent cells provided with the Gibson's assembly kit. Got a lot of colonies on the amp+ plate but when I isolated plasmid from them, I got no plasmid.
additionally, I ran the assembly reaction on the agarose gel, I was able to see fragments clearly. So how am I getting colonies if the assembly failed?
How to get rid of false positive colonies?
(Just to remind that I wanted to join three 4.1 kb fragments together so as to circularise them and make a plasmid like structure)
What could be the reason? Please help
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