Biomod/2011/TUM/TNT/LabbookA/2011/09/12

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12th Sep 2011

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Size Exclusion Filter Purification

Folded samples BM5_5/20, BM10_5 and BM11_20 were filtered in Amicon (100 kDa) with FOB20.

Sample Filtering

  • 100 µl from all three samples were mixed and put into a Amicon filter (100 kDa)
  • 400 µl 1x FOB20 added
  • spinned for 5 minutes at 14000 rcf at 4°C
  • 400 µl 1x FOB20 added
  • spinned for 5 minutes at 14000 rcf at 4°C
  • 400 µl 1x FOB20 added
  • spinned for 5 minutes at 14000 rcf at 4°C
  • 400 µl 1x FOB20 added
  • spinned for 5 minutes at 14000 rcf at 4°C
  • 400 µl 1x FOB20 added
  • spinned for 5 minutes at 14000 rcf at 4°C
  • Turn the filter upside down and spin the purified sample into a new Ependorf tube for 2 minutes at 1000 rcf

Results

  • Approximately 30 µL of filtered sample was obtained from each batch.
  • The spectrum of BM5_5/20 was measured using the nanodrop photometer and the peak at 260 nm was more than 5 times lower compared to the spectrum of an unfiltered sample. There are no extinction coefficients for folded DNA available so that DNA concentration can only be roughly estimated to be ca. 10 nM based on experience.
  • The concentration of fluorescence labels could not be determined by nanodrop as the concentrations are too low for this device.