So I always have this weird results during my gene cloning experiments. I perform restriction enzyme digestion (double digest) for my plasmid vector (7.0 kb) and insert gene (1.5 kb). I perform ligation using T4 DNA ligase, incubate for 16 h at 16 C, then E. coli DH5a transformation with proper controls.
After picking colonies, plasmid miniprep, I get pDNA transformants with (circular) size smaller than the control (so many of them). I always get this kind of results even in the past, I can't get proper transformants and one of the problem is this and the other is low transformation efficiency. I am not sure what is the problem? Can anyone help me?
To me it's a host problem, when I switch to E. coli HST08 from DH5a, I get better results and higher rate of successful transformants. But some colleagues said my DNA concentration is too low, although I've been using 150-200 ng of DNA vector and the 5:1, 10:1 or more insert:vector molar ratio even in the past. Any one with the same problem?
Hi Edward,
it sounds to me as if you have a plasmid contamination somewhere. I would recommend remaking all solutions you have used. You could also check that your enzymes haven't been contaminated by transforming a ul of them.
On the other points:
To me your DNA concentration is at the high end not the low end. For ligations and transformations less can be better - I think 10-50ng of vector to be a better value to use. Followed by 1ul ligation to 10ul cells (if high efficiency cells).
As for the strain, checking the transformation efficiency will indicate whether you have a problem there, you don't say if they are commercial or in house produced, but the higher the efficiency the better. Buying some library efficiency DH5a can be a good investment, as (for thermofisher's) they are around 100x more efficient. You can even use less per reaction and still get better results than the cloning efficiency ones.
-Richard
Hi Dr. Richard,
it sounds to me as if you have a plasmid contamination somewhere. I would recommend remaking all solutions you have used. You could also check that your enzymes haven't been contaminated by transforming a ul of them.
- I think I've ruled them out, since I always use negative controls [(1) no plasmid DNA, (2) vector single cut, (3) vector double cut and (4) recircularized vector)]. So reagents seems not the problem for me. I also use commercial E. coli DH5a from local source here.
On the other points:
To me your DNA concentration is at the high end not the low end. For ligations and transformations less can be better - I think 10-50ng of vector to be a better value to use. Followed by 1ul ligation to 10ul cells (if high efficiency cells).
- Will there be a problem if I use higher concentrations? Because I want to maximize my transformation efficiency and my vector is my own construction, not from commercial source. So 50 might be little low for my desired output. Even so I don't get higher TE using high concentration, how much more lower concentration pDNA vector? I also calculate my insert:vector molar ratio with increments of 5:1, 10:1 and even more for that case. However still I didn't get my desired transformants and pDNA.
As for the strain, checking the transformation efficiency will indicate whether you have a problem there, you don't say if they are commercial or in house produced, but the higher the efficiency the better. Buying some library efficiency DH5a can be a good investment, as (for thermofisher's) they are around 100x more efficient. You can even use less per reaction and still get better results than the cloning efficiency ones.
-- I don't think my commercial E. coli DH5a have problems although I am hypothesizing that there are some genotypic differences between strains. I don't know how much effect it has on my transformation experiments. Also taking into consideration the combination of RE cutters (SacI and XbaI) that I use as well as the toxicity of the insert gene.
However, I appreciate your answer. It makes me think over and over again and try to rule out the things that had been done and not yet done.
Hi Edward,
- Will there be a problem if I use higher concentrations?
my empirical observations is that there is a point at which using a higher concentration is counter productive.
- my vector is my own construction, not from commercial source.
Unless you have added some elements that reduce the plasmids ability to maintain itself then this shouldn't make any difference at all.
- So 50 might be little low for my desired output
but your desired output is a recombinant plasmid with your insert in. If using lower concentrations of DNA gives you that, then I don't see what your worry is here.
-I also calculate my insert:vector molar ratio with increments of 5:1, 10:1 and even more for that case.
As you are using a double digested vector you don't need such high ratio's, using v:i ratios of 1:1 and 1:3 would be much better - higher ratios can prevent ligation as insert will ligate together rather than with the vector.
- my commercial E. coli DH5a have problems although I am hypothesizing that there are some genotypic differences between strains. I don't know how much effect it has on my transformation experiments.
The genotype should be what is expected if they are a reputable company, although comparing them to a different source could be a good idea.
-Richard
Edit: one extra control you can run would be insert+ligase to test that there's nothing unwanted in the insert that can generate a replication competent plasmid.
Hi,
I want to believe that you cut your minipreps with a restriction enzyme to get the lineal form of the DNA? sometimes you can have several forms of supercoiled plasmid DNA that run longer than normal... and if you used different strains they can have different topoisomerases...
Other option if you want to avoid the cut & paste old system is the NEB assembly kit, with this you design 2 couples of overlapping primers, amplify the vector and the insert in two PCR reactions and then you assemble them, no need for restriction sites and you can insert your DNA wherever you want...
Good luck,
Alfonso
Hi Dr. Richard,
Thanks for your recommendations. I will look into it in more detail.
Hi Dr. Alfonso
I want to believe that you cut your minipreps with a restriction enzyme to get the lineal form of the DNA? sometimes you can have several forms of supercoiled plasmid DNA that run longer than normal... and if you used different strains they can have different topoisomerases
- I make sure I get 1 cut, by (1) calculating the right amount of DNA weight to be used for digestion. Usually, 1 ug per unit of enzyme for 1 hour digestion. (2) I take an aliquot and check the bands with control in agarose gel electrophoresis. (3) If there are still undigested, I will adjust and add more enzyme and extend the reaction time for a couple of hours more. (4) After I see almost 1 cut after hours of reaction, I will proceed to gel extraction. I believe I'm making sure or maximizing that there will be no supercoiled DNA.
Other option if you want to avoid the cut & paste old system is the NEB assembly kit, with this you design 2 couples of overlapping primers, amplify the vector and the insert in two PCR reactions and then you assemble them, no need for restriction sites and you can insert your DNA wherever you want.
- I will try to check this one out. Thank you!
Hi Edward,
I too face similar problem now. I try to clone 2.2 kb insert into 8 kb vector but after ligation and transformation I get 5-6 kb vector only. How did you sorted out your problem. I tried DH5 alpha, Top10 from various sources. Suggest me your modifications
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