Biomod/2011/TUM/TNT/LabbookA/2011/09/02

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       TUM NanU - Home   

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Folding Fluorophore labeled theU Structures

Overview

Labeled oligos for FRET bulk measurements should be inserted in the U structure. The structure was labeled with donor only, acceptor only and both donor and acceptor fluorophores. All these structures were folded. The structures are named BM3_4/29, BM4_8/23, BM5_5/20, BM6_4, BM7_29, BM8_8, BM9_23, BM10_5 and BM11_20. For details see: structure page and setting up folding reactions.

Preconsiderations

Concentrations of the fluorophore labeled oligos could only be determined roughly due to impurities in the absorption spectra. According to the spectra, concentrations vary from 2µM to 7µM. Therefore all concentration of all labeled oligos were assumed to be ca. 2µM. For each folding reaction, 20 nM scaffold (p7560) are used, and staples are added in tenfold excess, i.e. 200 nM. That matches 10 µl of each appropriate labeled oligo in a 100 µl reaction batch.

Working Stocks

The working stocks contain all but the labeled staples.
E.g. the working stock for BM3_4/29 contains the following prestocks:

  • core
  • helix 5
  • helix 7
  • helix 8
  • helix 20
  • helix 22
  • helix 23
  • the special prestocks helix 4 w/o 175 and helix 29 w/o 225

E.g.: Helix 4 has 8 staples which are suitedto attach fluorophores to the structure. These are combined in prestock helix 4. For the structure BM3_4/29, two special prestocks are mixed: prestocks helix 4 w/o 175 and helix 29 w/o 225
Prestocks helix 4 w/o 175 contains only 7 staples. That one, which is to be labeled (staple 175), is missing in this prestock and added later.

Folding Batches

Strip tube 1 contains all donor only and acceptor only folding batches, one batch each structure. Strip tube 2 contains five identical folding batches of each 100 µl for BM3_4/29. Accordingly, strip tube 3 contains five batches for BM4_8/23 and strip tube 4 five batches for BM5_5/20.

Each folding batch consists of

  • 20 µl 100 nM scaffold (p7560)
  • 40 µl of the appropriate working stock, containing all unlabeled staples at 500 nM
  • 10 µl 10x FOBxM (50 mM Tris, 10 mM EDTA)
  • 10 µl 200 mM MgCl2

For single labeled structures, i.e. all donor onyl and acceptor only structures, 10 µl of the labeled oligo were added. Then the batch was filled up with 10 µl ddH2O to a total volume of 100 µl.
For the three batches containing both donor and acceptor, 10 µl of each of the two labeled oligos were added without additional water.

Finally all batches were incubated in the thermocycler using the 15_65 folding ramp.