Biomod/2011/TUM/TNT/LabbookA/2011/08/23

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23th Aug 2011

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Folding first Structures: theU

Folding reactions for the structures BM1 and BM2 were prepared according to the general protocol. Both structures were folded once using the 15_56 thermal ramp, once with 2D_H3_ML and once with 5D_H3_ML.

Preparing DAPI Concentration Series for the Helix MH255/256

In all samples, helix concentration should be 10 nM.
Donor-only and acceptor-only samples are prepared for control measurements.
Buffer used: 0.5x TBE, 11mM MgCl2

Stock solutions of fluorophore labeled oligonucleotides are 2.8 µM (MH_255 Atto 647N ddUTP) resp. 7.1 µM (MH_256 Atto 550 ddCTP).

Donor only helix:

  • 1.4 µl MH_256 Atto 550 ddCTP
  • 0.1 µl unlabeled MH_255
  • 998.5 µl buffer


Acceptor only helix:

  • 3.6 µl MH_255 Atto 647N ddUTP
  • 0.1 µl unlabeled MH_255
  • 996.3 µl buffer


100 nM stock helix with donor and acceptor:

  • 7.1 µl MH_255 Atto 647N ddUTP
  • 2.8 µl MH_256 Atto 550 ddCTP
  • 190.1 µl buffer


DAPI is added in units of KD. The KD is 1.8 nM. Two stock solutions of DAPI were prepared, with concentrations of 188 µM and 9 µM. The following table shows the pipeting scheme as well as the expected amount of binding sites of the DNA occupied with DAPI.


Table 1: Pipeting scheme and expected amount of binding sites of the DNA occupied with DAPI.

used DAPI stock DAPI [µL] buffer [µL] labeled helix stock (100nM) [µL] [DAPI] in units of KD expected amount binding sites occupied with DAPI
180 µM 19 71 10 19 19/20
180 µM 9 81 10 9 9/10
180 µM 4 86 10 4 4/5
180 µM 2 88 10 2 1/3
180 µM 1 89 10 1 1/2
9 µM 10 80 10 1/2 1/3
9 µM 5 85 10 1/4 4/5
9 µM 2.2 87.8 10 1/9 1/10
9 µM 1.1 88.9 10 1/19 1/20


Samples were hybridized by incubating them in the thermocycler, starting at 65°C and cooling down by 1°C per minute for 35 minutes
Hybridized samples were stored at 4°C.