Science Buddies: "Ask an Expert"

Besides Gel Electrophoresis, are there other well know known and used methods for separating DNA fragments?

Thank you

Hi!

You can try recombinant DNA I belive but it appears to be pretty difficult. However, I am not sure about this. Try looking at this site:

1. http://users.rcn.com/jkimball.ma.ultran ... ntDNA.html

Hi there,

I'm not sure what Jessica means by recombinant DNA technology. In any case, from your previous posts I remember that you're amplifying a gene by PCR and cloning it into a vector.

I'm guessing that you're thinking about (or at the step) where you want to check that you amplified the correct sized product by PCR. Unfortunately, I do not know of any way to accurately assay this without performing gel electorphoresis. Do you not have access to this type of equipment? Or are you concerned with working with potential carcinogens like ethidium bromide?

If you post your concerns, we might be able to come up with other suggestions.

I'm sorry I can't be more helpful. I can't really think of other ways to verify the size and purify your product in one step. I think gel electrophoresis is the most cost-effective way to go.

Best of luck,
Linh

Hi

I take that back about recombinant DNA. I don't think it is possible. Sorry about that.

Hi,

I agree with Linh, gel electrophoresis is probably the best way. There are columns that can be used to separate larger pieces of DNA from the unreacted bases of the PCR and the Taq, but its usually best to check that the PCR product is the size you expect first. People often then use a similar type of column to extract the DNA from the slice of the gel with the correct sized product or there are other methods for extracting the DNA from the gel.

Did you find a PCR machine then? What did you decide to do about a vector? Or are you planning ahead?

-Caroline

Hi,

Besides gel electrophoresis, you can use anion exchange, diatomaceous earth or hydroxyapatite to purify a quantity of DNA. Here is a brief description of each method.

1. Anion exchange chromatography. You would need an anion exchange resin with a large pore size (to accommodate the DNA molecules). The resin is equilibrated with a neutral pH buffer and eluted with a high salt (NaCl) buffer. DNA of different sizes can be separated by gradually increasing the concentration of NaCl.

2. Diatomaceous earth: This is a method for purifying plasmids. The plasmid DNA is bound to diatomaceous earth in a high salt/high ethanol buffer and eluted in water.

3. Hydroxyapatite: This is a chromatography support that will bind DNA in 10 mM phosphate buffer at pH 6.8 and eluted with high phosphate buffer. Different sizes of DNA can be eluted by gradually increasing the concentration of phosphate.

Let me know if you need more information on any of these techniques.


Donna Hardy