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==thermal ramps==
==thermal ramps==
We tried different thermal ramps for folding our structures. The most simple one is called 15_65. Here the thermocycler starts at a temperature of 65°C. Every 15 minutes temperature is decreased by 1°C, until the final temperature of xxx °C is reached.
<b>XXX look up parameters of folding ramps </b>

Revision as of 01:16, 25 October 2011

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Setting up folding reactions

prestocks

Oligonucleotides are first mixed in prestocks. This are stock solutions of staples that are usually used together, e.g. all staples that form the core of the structure are mixed to a prestock, all staples that add polyT ends to the core are another prestock and so on. Mix 10µl 100µM of each staple for a prestock.

working stock

Working stocks are mixed from the appropriate prestocks. Different amounts of staples in each prestock are taken into account by adjusting the volumes of the prestocks. So if 109 staples are mixed in the core prestock, then 109µl of this prestock are used to mix the working stock. If the total volume of all prestocks mixed in this way is less than 200µl, then ddH2 is added up to a final volume of 200µl working stock.
For example, the working stock for the structure BM2 consists of:

  • 109µl prestock core
  • 8µl prestock helix 4
  • 7µl prestock helix 5
  • 7µl prestock helix 7
  • 7µl prestock helix 8
  • 7µl prestock helix 20
  • 7µl prestock helix 22
  • 7µl prestock helix 23
  • 8µl prestock helix 29
  • 33µl ddH2

folding batch

For unlabeled structures, a folding batch consists of:

  • 20µl scaffold (p7560)
  • 40µl Fob20, which in turn contains one equivalent of 10x FOBXM (50mM Tris, 10mM EDTA), one equivalent of 200mM MgCl2 and two eqivalents of ddH2O
  • 40µl working stock (i.e. all staples at 500nM concentration)

For structures containing fluorophores, the composotion might be adjusted according to the concentrations of the labeled oligonucleotides. See the appropriate entries.

thermal ramps

We tried different thermal ramps for folding our structures. The most simple one is called 15_65. Here the thermocycler starts at a temperature of 65°C. Every 15 minutes temperature is decreased by 1°C, until the final temperature of xxx °C is reached.

XXX look up parameters of folding ramps

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