Chromatography Forum

Hello all,

I am currently looking for a method to separate a mixture of DNA fragments, meanly of 2 and 4 kb lenght. Normally DNA lenght separation is done with gel electroforese, however I do know that small DNA fragments like primers can be purified with a HPLC. For us, gel elektroforese is not possible as we cannot PCR our sample, and therefore we want to explore the possibilities of using an HPLC. Additionally, HPLC will allow us to fractionate our sample, which makes further analysis possible.

Therefore I was wondering if anyone has any experience with this, or can point me into a direction. What type of column would be most usefull, size exclusion, ion-exchange, etc?
Do you know any method described. Etc

With kind regards,
Rutger

My recommendation is to start with aqueous SEC columns. Some manufacturer offer special designed DNA columns. Small particle size and maximum 300A poresize I would look for.
Good luck.

Also, anion-exchange works well for this. I can send you an example of a 20-base oligonucleotide being separated by nearly two peak widths from the same oligonucleotide with one additional base. Ask offline.

The same method works as well for double-stranded products from PCR reactions.

Andy Alpert
PolyLC Inc.
aalpert@polylc.com