Biomod/2011/TUM/TNT/LabbookA/Setting up folding reactionsng reactions
Setting up folding reactions
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. Also we made prestocks for all staples of one helix, which are suited to be labeled with fluorophores. Mix 10µl 100µM of each staple for a prestock.
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
For unlabeled structures, a folding batch consists of:
- 20µl scaffold (100nM)
- 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 (staple concentration 500nM)
So the batch contains 20nM scaffold and 200nM of each staple.
For structures containing fluorophores, the composotion might be adjusted according to the concentrations of the labeled oligonucleotides. See the appropriate labbook entries.
We tried different thermal ramps for folding our structures.
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.
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.
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.