Chromatography Forum

Hi everybody,

I am a brand new user and I need some help on an aspect of my project that I am having a great deal of difficulty with. I need to make pure dsDNA (54mer) to test a protein-DNA interaction. To make the dsDNA I anneal the two single stranded DNAs. However, some single stranded DNA still remains in the mixture. I have tried using IP-RP HPLC with tetrabutylammonium bromide as the ion-pairing reagent with adequate results but it is impossible to remove from the eluted product which interferes with my interaction studies. So instead of using IP-RP HPLC we have decided to use anion-exchange HPLC with a Waters Spherisorb SAX Column (porous, silica based). I was wondering if anyone had any suggestions of buffers to use for the SAX column? Any help would be greatly appreciated. Thanks.

Not my field, but maybe this article will help:

Sundaram, K., & Sloane, L. (1995). Liquid chromatographic assay for the separation of single- and double- stranded DNA by using UV and UV diode-array detectors and hydroxylapatite column. Journal of Liquid Chromatography, 18(5), 925-939. Retrieved from Biological Abstracts 1969 - Present database

Thanks for the suggestion, but I thought of using the hydroxylapatite column but our lab group doesn't have one and we really don't want to buy a column for a one time use. This is why I have opted to go with the SAX column but I am unsure of what eluent I should use.

Use 0.1 mol/L triethylammonium acetate as RP-IP reagent. It can be removed afterwards using a speed vac. You should be able to separate the ss from the ds

A: 0.1mol/L TEAA, pH = 7
B: 0.1mol/L TEAA, 25% acetonitrile
T=50°C

Adjust gradient accordingly.

I used an ion-exchange column [Waters Gen-Pak FAX, UV@260nm, 0.6 mL/min, 20°C] to study the annealing of a 26-mer ODN but the column temperature seemed to play a role in the ss/ds ratio. Temperatures >10°C seemed to cause the dsDNA to denature...at 20°C it denatured completely and all I see is ssDNA.

A: 150 mM TRIS at pH 8 with 15%MeCN
B: 150 mM TRIS at pH 8 with 15%MeCN + 1M NaCl

Your DNA is ~2X longer so you might not have that problem, but I vote for the TEAA ion-pair method!

Good luck, I'm interested to hear how things work for you.

K.