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        Oligonucleotide Purification

        互联网

        1412

        Solutions

        Gel Stocks

        Diluent 5X Buffer 25% Acrylamide

        209 g Urea 209 g Urea 209 g Urea

        up to 500 ml Q 250 ml 10X TBE 120.8 g Acrylamide

        up to 500 ml Q 4.1 g BIS

        up to 500 ml Q

        2.5 M NH4 OAc

        19.2 g NH4 OAc

        up to 100 ml Q

        Formamide Dye

        9 ml deionized formamide

        500 m l 10X TBE

        500 m l 0.2% bromophenyl blue and 0.2% xylene cyanol

        Other Reagents Needed:

        0.22 m M disposable syringe filters, short wave UV source, intensifying screen for UV shadowing

        Procedure

        • Pour a 20% denaturing gel: 12 ml 5X Buffer, 48 ml 25% acrylamide, 500 m l 10% APS (Gibco #15523-012), 20 m l TEMED (Sigma #T8133).

        • Dry down 100 m g oligonucleotide and resuspend in 50 m l Formamide Dye.

        • Boil for 2 minutes and hold on ice until ready to load gel.

        • Run the gel at 15 W constant power until the fast dye is at the bottom of the gel.

        • To visualize the bands, UV shadow on the intensifying screen. Excise the band and crush and soak in 1 ml 2.5 M Ammonium Acetate at 37°C for several hours to overnight.

        • Spin for 10 minutes and remove the supernatant. Add another 1 ml of 2.5 M ammonium acetate, pellet and pool with the previous eluate.

        • Filter the supernatant through a 0.22 m M filter, add 0.1 volumes of 3M NaOAc 5.2 and 2 volumes EtOH. I usually add 1 ml glycogen also.

        • Spin for 30 minutes, wash and dry. Resuspend in 50 m l TE and quantitate 5 ml.

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