Separation of dsDNA from ssDNA by HPLC

[Wazi]

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.

[bisnettrj2]

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

[Wazi]

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.

[rhaefe]

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.

[ksharp]

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.