Biomod/2011/TUM/TNT/LabbookA/Setting up folding reactionsng reactions: Difference between revisions

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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.  
We tried different thermal ramps for folding our structures.  
 
<h3>15_65</h3>
<b>XXX look up parameters of folding ramps </b>
This shortest and most simple folding ramp starts at 65°C. Every 15 minutes the temperature is decreased by 1°C, to a final temperature of 25°C. After finishing this ramp, temperature is set to 4°C.  
<h3>2D_H3_ML</h3>
Here again, the starting temperature is 65°C. Every 15 minutes the temperature is decreased by 1°C, to a final temperature of 57°C. The next part of the ramp, down to 26°C, proceeds with a cooling velocitiy of 1°C per 1:30 hours. After finishing this ramp, temperature is set to 4°C.  
<h3>5D_H3_ML</h3>
For this longest ramp, again the first part is cooling down from 65°C to 57°C with -1°C per 15 minutes. Then, the batch is cooled down with 1°C per 3 hours. Finally, temperature is set to 4°C.

Revision as of 08:50, 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 ddH2O is added up to a final volume of 200µl working stock. The final concentration of each staple in the working stock is 500nM
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 ddH2O

folding batch

For unlabeled structures, a folding batch consists of:

  • 20µl scaffold (p7560)
  • 40µl 10x 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

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.

15_65

This shortest and most simple folding ramp starts at 65°C. Every 15 minutes the temperature is decreased by 1°C, to a final temperature of 25°C. After finishing this ramp, temperature is set to 4°C.

2D_H3_ML

Here again, the starting temperature is 65°C. Every 15 minutes the temperature is decreased by 1°C, to a final temperature of 57°C. The next part of the ramp, down to 26°C, proceeds with a cooling velocitiy of 1°C per 1:30 hours. After finishing this ramp, temperature is set to 4°C.

5D_H3_ML

For this longest ramp, again the first part is cooling down from 65°C to 57°C with -1°C per 15 minutes. Then, the batch is cooled down with 1°C per 3 hours. Finally, temperature is set to 4°C.

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