Biomod/2011/TUM/TNT/LabbookA/2011/09/02: Difference between revisions

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For the three batches containing both donor and acceptor, 10µl of each of the two labeled oligos were added without additional water.<br>  
For the three batches containing both donor and acceptor, 10µl of each of the two labeled oligos were added without additional water.<br>  
<br>
<br>
Finally all batches were incubated in the thermocycler using the 65_15 folding ramp.  
Finally all batches were incubated in the thermocycler using the 15_65 folding ramp.  




Revision as of 02:05, 7 September 2011

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folding fluorophore-labeled the_U structures

overview

labeled oligos for FRET bulk measurements should be inserte in the_U structure. For all three suited FRET pairs, donor only, acceptor only and donor-acceptor 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. The details on these structures can be found on the structure page.

preconsiderations

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

working stocks

The working stocks contain all but the labeled staples.
E.g. working stock for BM3_4/29 contains the prestocks core, helix 5, 7, 8, 20, 22 and 23 as well as the special prestocks helix 4 w/o 175 and helix 29 w/o 225.

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 100µl 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

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

For single labeled structures, i.e. all donor onyl and acceptor only structures, 10µl of the labeled oligo were added. Then the batch was filled up with 10µl ddH2O to a total volume 0f 100µl.
For the three batches containing both donor and acceptor, 10µl 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.

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