Biomod/2011/TUM/TNT/LabbookA/Purification of origamis by size exclusion filtration: Difference between revisions

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* apply 100 µl sample into an Amicon filter (100 kDa)
* apply 100 µl sample into an Amicon filter (100 kDa)
* add 400 µl FOBxM + 20mM MgCl<sub>2</sub>
* add 400 µl FOB20
* spin down for 2 minutes at 14000 rcf at 4°C
* spin down for 2 minutes at 14000 rcf at 4°C
* repeat washing for three times
* repeat washing for three times
* add 400 µl FOBxM + 20mM MgCl<sub>2</sub>
* add 400 µl FOB20
* spin down for 5 minutes at 14000 rcf at 4°C
* spin down for 5 minutes at 14000 rcf at 4°C
* turn the filter and spin down the purified sample into a new Epi for 2 minutes at 1000 rcf
* turn the filter and spin down the purified sample into a new Epi for 2 minutes at 1000 rcf

Revision as of 13:36, 27 October 2011

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Purification of origamis by size exclusion filtration

The first folded origamis were purified by agarose gel electrophoresis. This method is well characterized, but takes several hours and a huge amount of origami is lost in the gel. Since our structure folds quite properly and almost no misfolded origamis can be seen, such a thorough purification is not necessary in our case. We just need to get rid of the unused staples. Therefore, on the 6th September we tested purification with Amicon size exclusion filters for the first time. The molecular weight cut off is 100 kDa, so staples are filtered out, while origamis pass the filter.
First experiments were quite successful, insofar as the loss due to the filter was considerably smaller as that due to electrophoresis. Later, a PhD student of our lab tested varying conditions to optimize purity and minimize loss of origamis. All filtrations after the 14th September were done according to the following protocol, which gave the best results (i.e. no superfluous staples, good yield) in Martys experiments:

  • apply 100 µl sample into an Amicon filter (100 kDa)
  • add 400 µl FOB20
  • spin down for 2 minutes at 14000 rcf at 4°C
  • repeat washing for three times
  • add 400 µl FOB20
  • spin down for 5 minutes at 14000 rcf at 4°C
  • turn the filter and spin down the purified sample into a new Epi for 2 minutes at 1000 rcf
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