Hey Brian,

I wouldn't be too concerned at this point. It may be that in the reactions with excess DNA, some of the enzyme is sticking to the DNA substrate, which could result in an upward shift/smear since the "sticky" complex would then be a higher molecular weight. If you are really worried, you could take your digestion products and clean them up (column/gel purify or however your normal cleanup procedure goes) and make an aliquot at the concentration you plan to use in your ligation or transformation or whatever your next step is...then just run a few microliters of that and see what it looks like.

I would just go for it.

Cheers,

Jeremy

It just means there's a lot of DNA, so much it is having problems "walking" on the agarose "frame". Just remember to calculate the proportion between DNA and Digestion enzymes accordingly, and you won't have problems. And if you need a nice figure for a paper or thesis, dilute your DNA and load just a fraction of what you are currently loading on your gel.

As a general rule, 50 - 200 ng DNA per lane would give you a rather sharp band.

Hi Brian,

Take OD 260 and load say 50-100ng on your gel. One more thing may be that your DNA may have been suspended in lesser volume of TE so try to dilute your DNA further. But otherwise the quality seems fine.

Also don't run your gel at high voltage. 80-100V is enough because high voltage also causes these dragging of DNA on agarose gel because of heat up of your gel and buffer

Looking at the more concentrated lanes, I'm not too sure. It looks more like there is various sizes of DNA there, and doesn't really look like a "dragged DNA sample" (too high voltage).

I don't usually calculate how much DNA I need for transformation, but looking at the gel of my PCR it's usually more than enough, even after purification.

Looking at your gel, I'd say the first 2 lanes already look good enough for transformation (in terms of concentration and specificity). But just for curiosity or on the safe side you could try to use samples from all 5 lanes for transformation.

I want to restate that there is the same amount of DNA in each of these lanes - so the sheer amount of DNA in a lane cannot explain the different appearance. The purpose of this experiment was to determine just how much plasmid I could expect do digest in a reaction so that I don't burn through my enzymes unnecessarily. I didn't see anything below the cut band, so I'm confident that even the heavily-loaded reactions went nearly to completion, but I am concerned that (as Jeremy suggests) I may be carrying enzyme over. I follow this digest with an ethanol precipitation to further concentrate my samples - I will run that and update this question shortly.

Hi Brian, are you only check your plasmid preparation with digestion for further trasforming experiment? Or you need to digest this plasmid to linearize and to perform a cloning experiment? Sorry for the questions but I do not understand why you digest so much plasmid DNA, and why you want to do what you write in your last reply.

For your question, even if you load 1ug for all your sample, you load same amout of samples that were digested in different way. More concentrated, more enzymes used, worst digested (It is not a good thing to consume enzymes, and to use the same digestion volume, if I read well, for all the samples).

I agree with Jeremy you do not have to worry; in the image your undigest plasmid and your first samples appear good.

In my opinion your last samples do not have a complete digestion, but only a partial one so you have a smear of (a lot) different species of plasmid that try to run and separated on a gel without much success!

Valeria -

The transformations I am doing require 5 ug of DNA in 5 ul or less, and linearized plasmid works better. All of these digests were done with two ul (20 units) of enzyme in 100 ul reactions - I did not use more enzyme, because the purpose was to see how much I could digest with a fixed amount of enzyme.

I have convinced myself that the last digestions are complete because I see no indication of the supercoiled plasmid evident in the undigested first lane. I don't understand how I could have "a lot of different species of plasmid" because I am only loading one plasmid, and cutting at a single site. The only options I see are undigested plasmid (first lane), correctly cut plasmid (second lane), or overdigested plasmid (which should appear smaller). Could you explain what you mean by different species of plasmid?

Hi Brian, I do not explain very well what I want to say, for different "species" of plasmid I only want to say that you have linearized and undigested plasmid all together.

I was concerned that you will need this digestion for a cloning experiment, but if you only need the linearized plasmid so there is no problem as I said before. I never used so much DNA for a transformation, (in my situation inhibits transformation) but probably you are in a different situation, I ask to know and to learn something new!

I only want to say that maybe would be better to use the 3or 5ug digestion because your have more clear result in the gel respect the last two digestions, in particular if you concentrates the samples, but you know better than me what you need!

Wish you great results!

Artur, I did load different volumes. I only partially understand your explanation. Could you explain how the "density" across the gel would change with how much volume I add? Each lane should have the same amount of DNA, I am essentially just adding a little more water/load dye. I can imagine how a larger volume might allow for some of the DNA to be higher up in the well ... is that what you mean?