I did several minipreps (different plasmids extracted from different cell lines) and when I run them on agarose gel 0.8% they don't look good (smear at high MW and it looks always like that).
The linear size of the plasmid is 5.25kb, hence I would expect to see the band of the supercoiled form around 2.9kb (it is never there). It looks always like in the picture (lane 3) while the same plasmid digested with 1 or 2 restriction enzymes shows bands of the correct sizes on the gel (lane 2).
I use Sigma GenElute Miniprep kit. I've always been careful to not go over 5 min of lysis incubation. I centrifuge for 2 min at max speed to remove ethanol. Elution with water.
A. What can be the cause of the poor quality of my preps? Could it be a problew with the kit I use?
B. If the double digestion shows bands of the expected size, can I still use the bigger fragment for a ligation, or is it way better to use bands cut out from digestions of high quality preps? And why is it better?
Hi there,
The HMW species you get are plasmid multimers due to either homologous recombination between plasmid molecules and/or lack of resolvase activity in charge of separating sister plasmid molecules (plasmid monomers are then physically linked) . Whatever the process involved, efficient digest will result in the expected linear product.
Hi everyone,
I'm also dealing with a Problem and I would like to ask about your opinion...
My Vector Backbone (same Plasmid; but cut from different regions for different genes) is varying 3.3kb to 3.4 kb and my inserts sequences are 2.1kb, 2.3kb and 2.3kb.
I tried with;
-1:2, 1:3 ratios,
-used fresh Buffers,
-didnt expose DNA sequences to UV too Long while cutting from gel,
-after isolating DNA sequences from gels, i run them to validate sequences and they were correct too,
-DNA concentrations (Vector Backbone) were changing between 9.5 to 11.5 ng/µl (and I increased Vector DNA amount with insert DNA amount regarding to 1:2 and 1:3 ratios too..)
-At the end of the Ligation, samples were incubated at 16C for overnight +/-18-20hrs
-agar plates were prepared fresh,
I would like to hear your recommendations & ideas..
Cheers,
Z. Ilker
It could also be that during your lysis and quenching steps in your prep that you are over mixing and shearing the gDNA leading to the smear. I generally mix very very gently by slowly inverting the tube while rotating. If the color doesn't disappear after one or two inversions. I let it sit for a few seconds
I don't think your DNA preps are that bad for minipreps, you have loaded a LOT of DNA on those lanes. If you were to load much less DNA then the background smears would disappear. While they are not ultra clean, I wouldn't worry too much about the quality of the preps. Dominique Liger has provided the most likely explanation of why the uncut is running with such a high MW size. It is perhaps a bit surprising but not entirely. If it worries you, then you can digest your plasmid with a single cutting enzyme and relegate at very low DNA concentration and retransform. You should get a number of clones with monomer form. If the E. coli strain you are using is recA- the form should stay true. If RecA+ then you will end up with a mixture of forms. But you don't need to do this if the digestions look correct to you.
I agree with Michael. The background is probably sheared genomic DNA. You always get some in a miniprep. If it really matters, cut out the band and elute it from the gel.
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