Biomod/2011/TUM/TNT/LabbookA/2011/10/20

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20th Oct 2011

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Investigation of Structures BM24 15_65, BM24 2D_H3_ML and BM25_4/29 15_65

The structures BM24 15_65, BM24 2D_H3_ML and BM25_4/29 15_65 were purified according to standard procedures.

Preparation of grids

Preparation of grids (box 2) according to standard procedures.

E1, E2, E3, E4 according to following scheme: pipet plan
E1: BM2 with 2.4 µM EtBr (corresponding to one molecule every 7bp)
E2: BM2 with 2.4 µM EtBr (corresponding to one molecule every 7bp)
E3: BM2 with 0.74 µM EtBr (corresponding to one molecule every 21bp)
E4: BM2 with 0.74 µM EtBr (corresponding to one molecule every 21bp)

E5, A6, A7, A8 according to following scheme: pipet plan
E5: BM2 with 432 nM DAPI (corresponding to one molecule every 7bp)
A6: BM2 with 432 nM DAPI (corresponding to one molecule every 7bp)
A7: BM2 with 144 nM DAPI (corresponding to one molecule every 21bp)
A8: BM2 with 144 nM DAPI (corresponding to one molecule every 21bp)

A9, A10, B6, B7, B8, B9
BM2 dilution: 1:25
A9: BM2 without any DNA binders
A10: BM2 without any DNA binders
B6: BM2 without any DNA binders
B7: BM2 without any DNA binders

B8: BM24 15_65 dilution 1:25 without any DNA binders
B9: BM24 2D_H3_ML dilution 1:25 without any DNA binders

Observed grids

Observed grids: D4 and D5 box 2

  • Stain was good
  • Dilution was good, but not homogeneous, structures showed a tendency to cluster together


Observed grid: B8 box 2

  • Stain was bad
  • Not enough structures, propose a dilution of 1:20


Observed grid: B9 box 2

  • Stain was bad, no structures found

TIRF

TIRF films of beads for calibration were taken.

took old bead sample, the films look ok I think

Preparation of TIRF slides

  • Control: structure in FOB20: looks fime.
  • Slides with 0,6µM and 2,0µM EtBr

both look bad, we need to redo those concetrations