Hello All,
I am currently struggling with getting a Gibson Assembly to work. I am trying to insert a 2.5 Kb insert into a roughly 12 Kb plasmid (modified pLEX-MCS). I have had the G.A work in the past with the same plasmid.
Currently, I am creating the insert with 25 BP of homology on both ends via PCR with Q5 polymerase, this is the same homology sequence I have used in the past. For the G.A itself I am using ~60 ng of the vector and ~40 ng of the vector which is 3:1 insert to vector. Incubating at 50 C for 60 minutes followed by transformation using 3uL of the product. Transformation of the G.A product, cut vector and insert result in no colonies while ~10ng of the uncut vector results in a field of bacteria on the plate.
Side note, the gene I am inserting is non-toxic to bacteria, I am PCRing the insert from a pBabe vector which does not seem to affect the bacteria containing it,
Thanks!
I'd tentatively blame your competent cells or transformation procedure. If transformation was top notch, and 100% of the transformed cells make it on the plate, you should always see a good amount of "background" colonies -- meaning ones containing carryover vector from PCR, incorrect assemblies and random products you can't make sense of.
The correct way to test your transformation process is to dilute a small test plasmid to 10 pg. And then that should give you a full plate, expressed as billions of colonies per ug, similar to the charts all the vendors show.
I've not had any success whatsoever with inserts containing introns, no idea why. Went back to conventional cloning as I do a lot of intron based cloning ...
Thank you for your suggestions. I went back to square one and re-purified my vector and insert and did a 4:1 insert to vector ratio and ended up with ~60 colonies. Using PCR I have found that 9 out of 10 colonies tested have my insert. I am going to chalk it up to older plasmid/ potentially degraded DNA.
Try doing electroporation of plasmid, next time? It might improve the transformation efficiency.
Dear Ulrike, I'm using the gibson assembly to assemble different fragments into a vector and some of the fragments contain intron sequences. Do you think this could be the reason why I never got properly assemble vectors (always vectors with wrong size)? i tried to change the molar ratios between inserts and vector from 1:1 to 3:1, used different bacterial strain mutated in RecA genes, I also tried the new HiFi DNA assembly kit from NEB but with no success.
Hi Paolo, do you have background at least?
I'm doing ca. 50 constructs a year and I've no time unfortunately to test a method extensively if it does not work quickly. I've tried Gibson 3 or 4 times on a plasmid-intron combination tweaking parameters and it never worked, I got background of course but no insertion.
I can't say if the Gibson assembly would have a general issue with ligation of introns as I did not test other pairs - but I know from my cloning work that there has always been trouble with intron ligation if you cut close to the donor/acceptor sites. Yielding to my ligation protocol for years now being changed to a cycling one (8 hours long cycling between 10C and 30C for 30 seconds each). That brings up the background often quite some but it yields me positive colonies .. Normal things like promoter and protein etc is compared to my trouble with introns insanely quick and simple, even worse are introns with pre-micro RNA's in them. So if you have problems I would recommend you play with the temperature profile either of your Gibson assembly or switch to regular restriction site cloning and use the cycling ligation. Make sure you heat inactivate the ligation (10' 70C), that seems to impact the transformation some reported.
And yes, I've seen once even with old-school ligation intron fragments (as I said, if cut close to the splice sites) delete partially in the oddest ways during ligation. Only way around was repairing by re-inserting the missing sequence. But make sure you don't have restrictions sites or homologies in which maybe a sequencing failure did not show previously. Re-Sequence the fragment to be sure!
Hi Ulrike,
thanks for your reply.
yes, I got many colonies and the plasmid isolated from them carry the vector backbone and one of the 3 fragment, but the other two that sould be flanking the former and carry the intronic sequences are lost or rearranged.
The intron-exon junction on the 2 flanking fragments are about 200bp far from the ends carrying the overlap sequence (20bp) involved in the gibson assembly process. Do you think they are close enough to mess up with the assembly?
I alreadt thought about switching to the classic restriction enzyme cloning, in this case the intron/exon junction will be 400 and 700 bp far from the restriction sites. Do you think it is going to work?
I'm sorry but I didn't get the point on the restriction site/homology regions. What is that about?
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