DMT On Purification of DNA Oligonucleotides <35mer Using Oasis HLB SPE Products

This application brief highlights on purification of DNA using Oasis HLB SPE products.

Capillary gel electrophoresis analysis of fractions from oligodeoxythymine (30-mer) SPE purification. 

0.1 M TEAA, pH 7 Buffer – commercially available For 100 mL of 0.36 M TEAA buffer:

Mix 94.5 mL of MilliQ water and 0.5 mL of glacial acetic acid. 

While mixing slowly add 5 mL of TEA, mix until it dissolves. 

pH of final 0.36 M solution is approximately 11.3 (desirable values are between 10.8-11.5)* 

* Keep in closed polypropylene bottle. Handle in hood, TEA has a strong odor. 

Troubleshooting

Flow rates of >0.5 mL/min in the load step (step 3) will cause sample breakthrough which reduces oligonucleotide recovery in final elution (step 7). 

Recovery Calculation

Recovery of target oligonucleotide is determined by analysis with a UV absorbance spectrometer. 

Take 10 µl of sample solution (prior to loading), dilute to 1mL and measure Absorbance A₂₆₀(L). 

Take 10 µL of final elution (step 7), dilute to 1 mL and measure absorbance A₂₆₀(E). 

V_E = elution volume from step 7 

V_L = elution volume from step 3 

Oligonucleotide Purity Determined by Capillary Gel Electrophoresis.

1. M. Gilar, E.S.P. Bouvier, J. Chromatography A, vol 890 (1), 167-177.