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)
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.