Biomod/2011/TUM/TNT/LabbookA/2011/09/06

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6th Sep 2011 <!-- sibboleth --><div id="m1" style="border:0px;"><div style="display:none;" id="id">m1</div><div style="display:none;" id="dtext"></div><div style="display:none;" id="page">Biomod/2011/TUM/TNT/LabbookA/20</div><div style="display:none;" id="fmt">yyyy/MM/dd</div><div style="display:none;" id="css">OWWNB</div><div style="display:none;" id="month">8</div><div style="display:none;" id="year">2011</div><div style="display:none;" id="readonly">Y</div></div> <!-- sibboleth --><div id="m2" style="border:0px;"><div style="display:none;" id="id">m2</div><div style="display:none;" id="dtext"></div><div style="display:none;" id="page">Biomod/2011/TUM/TNT/LabbookA/20</div><div style="display:none;" id="fmt">yyyy/MM/dd</div><div style="display:none;" id="css">OWWNB</div><div style="display:none;" id="month">9</div><div style="display:none;" id="year">2011</div><div style="display:none;" id="readonly">Y</div></div> <!-- sibboleth --><div id="m3" style="border:0px;"><div style="display:none;" id="id">m3</div><div style="display:none;" id="dtext"></div><div style="display:none;" id="page">Biomod/2011/TUM/TNT/LabbookA/20</div><div style="display:none;" id="fmt">yyyy/MM/dd</div><div style="display:none;" id="css">OWWNB</div><div style="display:none;" id="month">10</div><div style="display:none;" id="year">2011</div><div style="display:none;" id="readonly">Y</div></div>

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Size Exclusion Filter Purification of BM_2_15_65 and BM_2_5D_H3_ML

The folded samples BM_2_15_65 (2011/08/23) and BM_2_5D_H3_ML (2011/08/23) were filtered in Amicon filters (100 kDa) with 0.5 x TBE + 11 mM MgCl2

Sample Filtering

  • 50 µl from both samples were mixed and put into a Amicon filter (100 kDa)
  • 400 µl 0.5 x TBE + 11 mM MgCl2 added
  • spinned for 5 minutes at 14000 rcf at 4°C
  • 400 µl 0.5 x TBE + 11 mM MgCl2 added
  • spinned for 5 minutes at 14000 rcf at 4°C
  • 400 µl 0.5x TBE + 11 mM MgCl2 added
  • spinned for 5 minutes at 14000 rcf at 4°C
  • turned the filter and spinned the purified sample into a new Ependorf tube for 2 minutes at 1000 rcf
  • 1 x and 2 x filtered buffer filled into separate Eppendorf tube (the buffer, which came through the filter; if purification with spin columns works, this should only contain staples)
  • 3 x timed filtered buffer discarded

Control of Filtration Outcome

Figure 1 shows a 50 ml 2% Agarose-Gel with EtBr

Purification test 1 6 9 2011.jpg

Figure 1: From left to right: 1kb ladder, p7560, filtered BM_2, BM_2_15_65, BM_2_5D_H3_ML, 1st flowthrough, 2nd flowthrough

  • Residual staples seem to remain in the solution. Also the leading band is slower than the scaffold. Marty said this is normal, when purified samples are put on a gel.
  • We should make a new purification with less initial concentration and folding buffer as washing buffer
  • Look at samples in the TEM as soon as possible! (should be repaired tommorow)

Size Exclusion Filter Purification of folded BM_1_15_65

The folded sample BM_1_1565(2011/08/23) was filtered in Amicon (100 kDa) with 1 x FOB20

Sample Filtering

  • 50 µl sample in a Amicon filter (100 kDa)
  • 400 µl 1xFOB20 added
  • spinned for 5 minutes at 14000 rcf
  • 400 µl 1xFOB20 added
  • spinned for 5 minutes at 14000 rcf
  • 400 µl 1xFOB20 added
  • spinned for 5 minutes at 14000 rcf
  • 400 µl 1xFOB20 added
  • spinned for 5 minutes at 14000 rcf
  • 400 µl 1xFOB20 added
  • spinned for 5 minutes at 14000 rcf
  • turned the filter and spinned the purified sample into a new Eppendorf tube for 2 minutes at 1000 rcf
  • flowthroughs of all washing steps stored (if purification with spin columns works, this should only contain staples)

Control of Filtration Outcome

Figure 2 shows a 125 ml 2% Agarose-Gel with EtBr.

File:110906 filter test biomod emf-unpur emf-gel-buff-pur-fs58-1 emf-fob20-pur-fs58-2 1xfilt-through 7.modified2.tif

Figure 2: 1kb ladder, p7560, BM_1_15_65 5x filtered, BM_1_1565 unfiltered, 1. flowthrough, 2. flowthrough, 3. flowthrough, 4. flowthrough, 5. flowthrough, other samples: Martys stuff

Conclusion

After 5 times filtering, no more staples can be seen in the agarose gel, the samples can be considered pure enough for further analysis. Some origamis are lost due to the filtering, but this loss still seems to be smaller than that when purifying with preparative agarose gel electrophoresis.
To sum it up, this filtering procedure results in purified samples with higher amounts of origami and also works faster than the purification via agarose gel electrophoresis.

Photometric Spectroscopy of MH_255_647 and MH_256_550 with EtBr

This experiment is done to compare the results from the real time PCR experiments and photometric spectroscopy.

Sample Preparation

Preliminary Considerations

  • Testing of DNA with and without labels
  • With label: 2x50 µl
  • Without label: 1x50 µl
  • Target concentrations for EtBr: 0, 0.044, 0.4, 7.4 (in Units of KD)

Stocks

  • Buffer: 0.5 TE + 11 mM MgCl2
  • 200 µM EtBr: 1.58 µl EtBr (25 mM) + 198.4 µl Buffer
  • 50 µM EtBr: 25 µl EtBr (200 µM) + 75 µl Buffer
  • 280 nM MH_255_647N
  • 710 nM MH_256_550


Labeled DNA

Concentration EtBr (in Units of KD) EtBr Stock [µL] MH_255_647N 280 nM [µL] MH_256_550 710 nM [µL] Buffer (0,5 TBE, 11 mM MgCl2)
0 0 3,6 1,4 95
0.044 1,07 (50 µM stock) 3,6 1,4 93,93
0.4 9,6 (50 µM stock) 3,6 1,4 85,4
7.6 45.6 (200 µM stock) 3,6 1,4 49,4


Control: Unlabled DNA

Concentration EtBr (in Units of KD) EtBr Stock [µL] MH_255 280 nM [µL] MH_256 710 nM [µL] Buffer (0,5 TBE, 11 mM MgCl2)
0 0 0.5 0.5 49
0.044 0.54 (50 µM stock) 0.5 0.5 48.47
0.4 4.8 (50 µM stock) 0.5 0.5 44.2
7.6 22.8 (200 µM stock) 0.5 0.5 26.2

Hybridisation:

  • Incubation for 2 min at 65 °C
  • Decrease temperature 1 °C/min
  • Incubate for 1 min at 25 °C
  • Incubate for 1 min at 20 °C

Results

The experiments done with the spectrometer failed. No signal was detectable at all. The concentrations of the samples were below the detection limit. Since the concentration of the origamis will be even lower this method is not usable for bulk measurements.