DOC

Glycosylase assay

By Bernice Holmes,2014-05-06 12:24
17 views 0
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.

Report this document

For any questions or suggestions please email
cust-service@docsford.com