sepration of DNA on water

[asaroj27]

is it possible that we can isolate DNA on water instead of gel?

[MedGen]

Well there's a reason agarose and polyacrylamide are used for separating DNA. The molecules form cross links when setting that form an intricate lattice work throughout the gel. Depending on the concentration of gel solute used (agarose normally for DNA) defines the size of the lattice and how big the gaps are. The higher the concentration the more crosslinks form, the tighter the lattice, the better the separation between similarly sized bands.

 

Separating in water would just make them all migrate to the cathode at the same time, no separation. You need a medium that will retard the migration of different sized molecules, hence the whole point of using gel for electrophoresis.

[ecoli]

http://www.smarterthanthat.com/experiments/dna-madness/

 

You don't need a gel to isolate DNA... but its the best analytical way to do it.

[jimmydasaint]

Gels are probably very convenient but you can also use column chromatography for isolation of DNA:

 

A Sephadex column procedure for DNA isolation is also useful for detecting dsRNA

Alberto B.Livore, Peter Grubb', Clint W.Magill and Jane M.Magillt

Departments of Plant Pathology and Microbiology, 'Biochemistry and Biophysics, Texas Agricultural Experiment

Station, Texas A&M University, College Station, TX 77843, USA

Submitted November 18, 1987

 

Agarose gel electrophoresis of DNA isolated from certain strains of

Exserohilum turcicum (formerly Helminthosporium turcicum) gave 6 extra

ethidium stained bands bel ow the high mol ecul ar weight band of undigested

DNA. As the extra bands were not digested by restriction endonucl eases,

further experiments were undertaken to determine the nature of the bands. The

bands did not appear if extracts were first treated with RNase A which had

been treated to el iminate DNase (1). Further, the bands when stained with

acridine orange (2) fluoresced green on UV exposure, while low

molecular weight material fluoresced red. The material in the

bbaands could also be recovered by binding to cellulose fibers

* (Whatman CF-11) in the presence of 15% ethanol (3). These

resul ts l ead to the concl usion that the bands (arrows in E.t.

lane) are double stranded RNA. The occurrence of dsRNA

mycoviruses or of dsRNA inclusions in fungi where viruses cannot

be detected is quite common (4). In some cases the number and

size of the dsRNA species present can be used to identify

specific fungal strains and may be related to pathogenicity (5).

In other cases, including E. turcicum, the existence of dsRNA

inclusions has been implicated by serological techniques, though

no dsRNA has been isolated (6). We feel that the simplicity and

efficiency .of the procedure will make it extremely useful to

others interested in locating and characterizing dsRNA from

fungi and perhaps from other organisms as well especially when

isolation of DNA is also desirable.

http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=334703&blobtype=pdf

[MedGen]

Very confusing, the thread title says separation, but in the OP it says isolate. Gel electrophoresis isn't really used for isolation from other cellular fractions, which is what I thought the OP was asking about.

[jimmydasaint]

From the OP it seems that the question is more about isolation of DNA so I wanted to suggest a method that involved an 'aqueous' aspect to it. It is not quite clear though.

[MedGen]

Isn't Sephadex just the type of cationic bead used in the column for size exclusion chromatography though. Gel electrophoresis technically has an aqueous nature, the running buffer, the solution of DNA and the running dye. So it is just a form of chromatography as well in a way. The gel is the stationary phase and the buffer/DNA solution is the mobile phase, and its aqueous.

 

I need some clarification as to whether this is meant to be isolation or separation, hello OP? Hello?

[asaroj27]

hello sir

i am apologise for my mistake.actually i want to know that sepration of DNA on water

thanks for correction

[CharonY]

Separation according to size or from other molecules?

[asaroj27]

hello sir

according to basis of size or by any mean.

[CharonY]

Sorry, this does not make it really clear to me. Size separation are usually done in a matrix of whatever kind to exploit the morphological or weight differences (as DNA of different sizes do not differ in their overall chemical properties). Another method is for instance to exploit dielectric properties of DNA. While it does not require a matrix per se, one has to create areas of different electric potentials.

 

Using e.g. silica columns only allows a very rough separation, that is, everything below a certain cut-off will not be retained within the filter.

 

All of this can be done in an aqueous solution (though a certain amount of ions are needed), without an actual agarose gel.

[jimmydasaint]

Correct. Silica columns have a cut off of 400-500 bp, if I remember correctly. However, there is another method I have found after a bit of searching:

 

 

Microchip electrophoresis has become a mature separation technique in recent years. Compared to agarose gel electrophoresis, which is commonly used for DNA separation, microchip electrophoresis has several advantages such as automation, fast analysis speed and minimum sample requirement. For the fabrication of electrophoretic microchips, silica-based and polymer-based materials are two commonly used substrates. Among the polymer-based materials, poly(methyl methacrylate) (PMMA) substrate can be wire-imprinted in a common laboratory to form microfluidic channels without expensive fabrication facilities. Moreover, the neutral hydrophilic surface chemistry of PMMA allows direct DNA separation to be performed on bare microchips without the tedious surface modifications that are normally required for silica-based materials. This chapter presents an imprinting method for fabricating PMMA microchips as well as the on-chip assay for performing electrophoretic DNA separation on the fabricated microchip.

(the emphasis is my own).

http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-426-1_1

 

Is this what you were looking for asaroj27?

[CharonY]

Actually the cut-off of silica columns can be tweaked. Regarding the microchips, what is emphasized just means that the material itself is hydrophilic. All microchip based DNA separation require a hydrophilic surface. This can be a property of the material, or you have to treat the surface (e.g. with oxygen plasma).

In any case again, you require something in the channels (e.g. linear acrylamide, a sieving matrix, whatever) to allow for size separation. In other words, you employ similar techniques (with few exceptions as dielectrophoresis) in microchips as in conventional DNA electrophoresis. Advantages are mostly speed and sample consumption.

[jimmydasaint]

I was actually emphasising the neutral surface because the OP wanted something close to good ole H2O without modifications but thanks for the information. I can imagine that the separating channels require a polymer or sieving matrix as you have mentioned. I have learnt something new again. Thanks Charon Y.