Glycosylase assay

By Bernice Holmes,2014-05-06 12:24
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Glycosylase assay

Glycosylase assay

1ml 10x nicking buffer:

    500ul 1M Tris pH8 0.5M

    10ul 1M DTT 10mM

    20ul 0.5M EDTA 10mM

    100ul 10mg/ml BSA 1mg/ml

    370ul HO 2

    dispense 150ul aliquots and store at 20C

1ml 2x formamide loading buffer:

    100ul 10x TBE 1x

    900ul deionised formamide 90%

    5mg bpb/xc/og 0.5%

Use og only in large gel samples, no dye in small gel samples, and bpb/xc (v fluorescent)

    only in marker lanes.

Quantitate protein by Bradford assay and then by SDS PAGE. Use a sufficient amount of

    protein to give a visible product.

20ul glycosylase reaction:

    2ul 10x nicking buffer

    4ul annealed oligos (100nM)

    4ul protein

    10ul H0 2

Catherine used 10-50ul total volume

    Brian: 50nM substrate, 50nM protein for 1h. Catherine: 200ng protein (about 250nM).

Incubate at 37C for 30m.

    Add NaOH to 90mM and incubate at 90C in PCR block for 30m (DNA engine>MAIN>90C30M).

Precipitate a 50ul glycosylase reaction if a v clear picture is required. However,

    precipitation makes it more difficult to achieve fully denaturing conditions during PAGE.

Add 0.5ul 10mg/ml tRNA or ? glycogen th1/10 volume 3M NaOAc pH5.2

    3 volumes EtOH (-20C)

Precipitate at 20C for 1h to o/n.

Centrifuge at full speed for 20m.

Wash with 200ul 80% EtOH (-20C).

Centrifuge at full speed for 10m.

Resuspend in 10ul 2x formamide loading buffer, vortex and incubate for 5m at 99C.

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