Biomod/2011/TUM/TNT/LabbookA/2011/10/10: Difference between revisions

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* c<sub>EtBr</sub> = 0.69 µM,    V<sub>EtBr</sub> (@ 5 µM) = 5.5 µl
* c<sub>EtBr</sub> = 0.69 µM,    V<sub>EtBr</sub> (@ 5 µM) = 5.5 µl
* V<sub>FOBxM + 20mM MgCl<sub>2</sub></sub> = 33.5 µl
* V<sub>FOBxM + 20mM MgCl<sub>2</sub></sub> = 33.5 µl
* V<sub>tot</sub> = 40 µl
* Vortexed sample for 10 sec and shortly centrifuged it
* Let incubate for 5 min before making grids
===Sample Biomod_2_C3, Biomod_2_C4, Biomod_2_C5===
* controls without any DNA binder
* c<sub>BM_2</sub> ≈ 0.25 nM,    V<sub>BM_2</sub> (@ ≈ 10 nM) = 1 µl
* V<sub>FOBxM + 20mM MgCl<sub>2</sub></sub> = 39 µl
* V<sub>tot</sub> = 40 µl
* V<sub>tot</sub> = 40 µl
* Vortexed sample for 10 sec and shortly centrifuged it
* Vortexed sample for 10 sec and shortly centrifuged it

Revision as of 07:54, 11 October 2011

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TEM measurements of BM_2 with EtBr

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_B2, Biomod_2_B3, Biomod_2_B4

  • in equilibrium each 7 base pairs one EtBr molecule should have bound to the structure
  • All dilutions are made with FOBXM + 20 mM MgCl2
  • cBM_2 ≈ 0.25 nM, VBM_2 (@ ≈ 10 nM) = 1 µl
  • cEtBr = 2.27 µM, VEtBr (@ 5 µM) = 18.6 µl
  • VFOBxM + 20mM MgCl2 = 20.9 µl
  • Vtot = 40 µl
  • Vortexed sample for 10 sec and shortly centrifuged it
  • Let incubate for 5 min before making grids

Sample Biomod_2_B5, Biomod_2_C1, Biomod_2_C2

  • in equilibrium each 21 base pairs one EtBr molecule should have bound to the structure
  • All dilutions are made with FOBXM + 20 mM MgCl2
  • cBM_2 ≈ 0.25 nM, VBM_2 (@ ≈ 10 nM) = 1 µl
  • cEtBr = 0.69 µM, VEtBr (@ 5 µM) = 5.5 µl
  • VFOBxM + 20mM MgCl2 = 33.5 µl
  • Vtot = 40 µl
  • Vortexed sample for 10 sec and shortly centrifuged it
  • Let incubate for 5 min before making grids

Sample Biomod_2_C3, Biomod_2_C4, Biomod_2_C5

  • controls without any DNA binder
  • cBM_2 ≈ 0.25 nM, VBM_2 (@ ≈ 10 nM) = 1 µl
  • VFOBxM + 20mM MgCl2 = 39 µl
  • Vtot = 40 µl
  • Vortexed sample for 10 sec and shortly centrifuged it
  • Let incubate for 5 min before making grids

Experiment


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