Biomod/2011/TUM/TNT/LabbookA/2011/09/06: Difference between revisions
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<h2>Size exclusion filter purification of BM_2_15_65 and BM_2_5D_H3_ML </h2> | <h2>Size exclusion filter purification of BM_2_15_65 and BM_2_5D_H3_ML </h2> | ||
The folded samples BM_2_15_65 [http://openwetware.org/wiki/Biomod/2011/TUM/TNT/LabbookA/2011/08/23 (2011/08/23)] and BM_2_5D_H3_ML [http://openwetware.org/wiki/Biomod/2011/TUM/TNT/LabbookA/2011/08/23 (2011/08/23)] were filtered in Amicon filters (100 kDa) with 0.5 x TBE + 11 mM MgCl<sub>2</sub> | |||
<h3>Sample filtering</h3> | <h3>Sample filtering</h3> | ||
Line 43: | Line 43: | ||
<h3>Control of filtration outcome</h3> | <h3>Control of filtration outcome</h3> | ||
Figure | Figure 1 shows a 50 ml 2% Agarose-Gel with EtBr | ||
<center>[[Image: purification_test_1_6_9_2011.jpg]]</center> | <center>[[Image: purification_test_1_6_9_2011.jpg]]</center> | ||
<font size="1">'''Figure | <font size="1">'''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'''</font> | ||
* 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. | * 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) | * Look at samples in the TEM as soon as possible! (should be repaired tommorow) | ||
<h2>Size exclusion filter purification of folded BM_1_15_65</h2> | <h2>Size exclusion filter purification of folded BM_1_15_65</h2> | ||
The folded sample BM_1_1565[http://openwetware.org/wiki/Biomod/2011/TUM/TNT/LabbookA/2011/08/23 (2011/08/23)] was filtered in Amicon (100 kDa) with 1 x FOB20 | |||
<h3>Sample filtering</h3> | <h3>Sample filtering</h3> | ||
* 50 µl sample in a Amicon filter (100 kDa) | * 50 µl sample in a Amicon filter (100 kDa) | ||
* 400 µl | * 400 µl 1xFOB20 added | ||
* spinned for 5 minutes at 14000 rcf | * spinned for 5 minutes at 14000 rcf | ||
* 400 µl | * 400 µl 1xFOB20 added | ||
* spinned for 5 minutes at 14000 rcf | * spinned for 5 minutes at 14000 rcf | ||
* 400 µl | * 400 µl 1xFOB20 added | ||
* spinned for 5 minutes at 14000 rcf | * spinned for 5 minutes at 14000 rcf | ||
* 400 µl | * 400 µl 1xFOB20 added | ||
* spinned for 5 minutes at 14000 rcf | * spinned for 5 minutes at 14000 rcf | ||
* 400 µl | * 400 µl 1xFOB20 added | ||
* spinned for 5 minutes at 14000 rcf | * 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 | * turned the filter and spinned the purified sample into a new Eppendorf tube for 2 minutes at 1000 rcf | ||
Line 73: | Line 73: | ||
<h3>Control of filtration outcome </h3> | <h3>Control of filtration outcome </h3> | ||
Figure | Figure 2 shows a 125 ml 2% Agarose-Gel with EtBr | ||
<center>[[image:110906_filter_test_biomod_emf-unpur_emf-gel-buff-pur-fs58-1_emf-fob20-pur-fs58-2_1xfilt-through_7.modified2.tif]]</center> | <center>[[image:110906_filter_test_biomod_emf-unpur_emf-gel-buff-pur-fs58-1_emf-fob20-pur-fs58-2_1xfilt-through_7.modified2.tif]]</center> | ||
<font size=1>'''Figure | <font size=1>'''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'''</font> | ||
<h2>Conclusion</h2> | <h2>Conclusion</h2> | ||
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. <br> | 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. <br> | ||
To sum it up, this filtering procedure results in purified samples with higher amounts of origami and also works faster than the purification via 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. | ||
<h2>Photometric spectroscopy of MH_255_647 and MH_256_550 with EtBr</h2> | <h2>Photometric spectroscopy of MH_255_647 and MH_256_550 with EtBr</h2> |
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Main project page
Size exclusion filter purification of BM_2_15_65 and BM_2_5D_H3_MLThe 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
Control of filtration outcomeFigure 1 shows a 50 ml 2% Agarose-Gel with EtBr 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
Size exclusion filter purification of folded BM_1_15_65The folded sample BM_1_1565(2011/08/23) was filtered in Amicon (100 kDa) with 1 x FOB20 Sample filtering
Control of filtration outcomeFigure 2 shows a 125 ml 2% Agarose-Gel with EtBr 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 ConclusionAfter 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. Photometric spectroscopy of MH_255_647 and MH_256_550 with EtBrThis experiment is done to compare the results from the real time PCR experiments and photometric spectroscopy. Sample preparationPreliminary considerations
stocks
Labeled DNA
Control: Unlabled DNA
Hybridisation:
ResultsThe 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. |