Biomod/2011/TUM/TNT/LabbookA/2011/10/05

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5th Oct 2011

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Contents

TEM measurements of BM2 with Spermine

Intention

  • To get a feeling for how much base pairs per structure are occupied, we decided to prepare new grids for the TEM. Therefore we calculated the occupancy of the structures in a new way (see Calculation of intercalator concentrations) to get correct values.
  • We decided to prepare two samples and from each three grids.

Sample Preparation

Sample: Biomod_2_A1, Biomod_2_A2, Biomod_2_A3

  • In equilibrium each 7 base pairs one spermine molecule should have bound to the structure
  • All dilutions are made with FOB20
  • cBM_2 ≈ 0.5 nM, VBM_2 (@ ≈ 10 nM) = 1 µl
  • cSpermin = 1.34 µM, VSpermin (@ 2 µM) = 13.4 µl
  • VFOB20 = 5.6 µl
  • Vtot = 20 µl
  • Vortexed sample for 10 sec and shortly centrifuged it
  • Incubate for 5 min before making grids

Sample: Biomod_2_A4, Biomod_2_A5, Biomod_2_B1

  • In equilibrium each 21 base pairs one spermine molecule should have bound to the structure
  • All dilutions are made with FOB20
  • cBM_2 ≈ 0.5 nM, VBM_2 (@ ≈ 10 nM) = 1 µl
  • cSpermin = 0.42 µM, VSpermin (@ 2 µM) = 4.2 µl
  • VFOB20 = 14.8 µl
  • Vtot = 20 µl
  • Vortexed sample for 10 sec and shortly centrifuged it
  • Incubate for 5 min before making grids

Experiment

More structures

  • On purpose to see the influence of twist on our structure, we designed three additional structures that incorporate internal twist by adding base pairs each 21 bzw. 42 base pairs in the hole structure, one additional base pair per 42 base pairs just in the arms or just in the base of The U and in addition a structure with 100 randomly distributed additional base pairs all over the structure
  • the_U_v02_19_biotinadapter_42++
  • the_U_v02_19_biotinadapter_42_arms
  • the_U_v02_19_biotinadapter_42_base
  • the_U_v02_19_biotinadapter_21++ (just cando)
  • the_U_v02_19_biotinadapter_random100 (just cando)

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