Good morning,
after some time, I'm doing again some cloning. This time I decided to try the DNA assembly to simplify things (how naive). However, at about that time I re-discovered this paper
https://www.jbc.org/article/S0021-9258(20)33772-8/fulltext?sid=c047cc2b-3d2a-4182-8ed1-f82fd52b2110
so I wanted to simplify things even more and tried in vivo DNA assembly.
I’ll try to describe my protocol. The experiments in the beginning and lately differed slightly, so hopefully it will be understandable.
1) After isolation of my pENTR3C plasmid, I do restriction with EcoRV and SalI (both HF versions) in CutSmart buffer.
Lately, as I seemed to have plenty of uncut vector in my colonies – more on that later – I included also EcoRI-HF as killer cut, which cuts inside the part, which I want to cut out.
I always do gel electrophoresis to make sure the digest is complete. For example on attached file 220517_002, you can see results of triple (SalI+EcoRV+EcoRI), double (SalI+EcoRV) and single digests after 1, 2 and 3 hrs (I’ve put the double digest after 2hrs by error into well with 1hr sample, hence the empty line). Although the SalI enzyme seems to be less efficient. After 3 hours, if there is any uncut plasmid left, it’s barely detectable. Bigger problem could be leftovers of linearized vector.
2) For my insert, I do PCR with primers with 20 bp homologous to my vector.
Lately, as I used plasmid as template, I treated the PCR reaction with DpnI at 37°C for 1 hr.
3) I purify both plasmid and insert through columns. I don’t cut plasmid from gel, as I don’t have good experience with it, so there is the little cut-out piece purified as well.
4) Lastly, I mix the plasmid and insert in 1:3 to 1:10 molar ratio and transform into chemically competent E. coli DH5α cells (30 mins on ice with DNA; 30 s at 42°C; 2 min on ice; +500 ul of 37°C SOC and shaking at 37°C for 1.5 hr, I spread 20 and 200 ul on plates).
What is likely relevant is, that the cells are made in lab and they should have transformation efficiency around 10^7 CFU, which is rather low.
Regarding my results, I have two problems:
1) I noticed since my first experiments, that the vector-only control has more colonies than constructs, which should’ve worked, because I had plenty of insert DNA (constructs, where I had only little DNA, the number of colonies was similar to plasmid-only control). Thus, lately, I did experiment, where I varied amount of vector and insert DNA. Not surprisingly, the more plasmid, the more colonies. But surprisingly, the more insert I added, the fewer colonies I got. BTW all of them were uncut vector regardless.
How could that be possible? Am I doing something wrong?
2) during my last few attempts with cloning, most of my clones were empty. Last time when I did only the double digest, I got only empty vector. But based on the size of amplicon in colony PCR, it’s uncut (or linearized and re-ligated). When I did the triple digest, I got much fewer colonies (I went from 180 to 6, when treated with DpnI). Of these, 3 seem to be vector recombined at the attR sites lacking the whole Gateway casette, 2 uncut vector and 1 is my desired clone (based on sequencing with 1 primer, I need to sequence the rest, as I had no signal on colony PCR).
When I transformed triple digested plasmid with insert not treated with DpnI, I got 22 colonies. I tested 9 of them by colony PCR and sequenced 6 of them. Based on this, 1 is recombination product at attR sites, 5 are uncut vector, 2 are original vector and 1 is my desired product.
With double digested plasmid I got always 150+ colonies of each, I tested 9 by colony PCR and they all seem the same – uncut plasmid (as confirmed for 2 of them).
What could be the problem with my in vivo DNA assembly?
As I’m writing it now, what could seem as plausible explanation would be that majority of transformed plasmid is linearized version and if insert is taken up by bacteria as well, it may ligate only on one side, but not the other, so the bacteria ultimately doesn’t have plasmid, i.e. resistance and doesn’t grow, while bacteria with only the linearized plasmid (no insert) can re-ligate and grow happily ever after.
However, during the first attempts, it worked so so (50-30% of positive clones, which isn’t as high as e.g. here https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0137466, but still better than the latest results). The plasmid before transformation looked about the same on gel electrophoresis before and now. I still see the majority of plasmid as double digested (not linearized), so I don’t see, why should it now behave differently.
I don’t have any other explanation and as I don’t have any explanation, I have no idea, how to improve my in vivo DNA assembly efficiency.
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