Biomod/2011/TUM/TNT/LabbookA/2011/09/06: Difference between revisions

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===Ran a 125 ml 2% Agarose-Gel with EtBr===
===Ran a 125 ml 2% Agarose-Gel with EtBr===
=Photometric spectroscopy of MH255_647 and MH256_550 adding ErBr=
To compare results with RTPCR
==sample preparation==
===preliminary considerations===
*DNA with and without labels
*with Label: 2x50µl
*without label 1x50µl
*Target Concentrations for EtBr: 0, 0.044, 0.4, 7.4 (in Units of Kd)
===stocks===
*Buffer: 0.5 TE + 11mM MgCl<sub>2</sub>
*200 µM EtBr: 1.58µl EtBr (25mM) + 198.4µl Buffer
*50 µM EtBr: 25µl EtBr (200µM) + 75µl Buffer
*280nM MH255 647N
*710nM MH256 550
=== samples ===




Revision as of 05:55, 6 September 2011

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Size exclusion filter purification

Folded samples BM_2_1565 (23.8.2011) and BM_2_5Dh3ml (23.8.2011) filtered in Amicon (100 kDa) with 0.5 TBE + 11mM MgCl2

Sample filtering

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

Ran a 50 ml 2% Agarose-Gel with EtBr

  • 1kb ladder
  • p7560
  • filtered BM_2
  • BM_2_1565
  • BM_2_5Dh3ml
  • 1x filtered buffer
  • 2x filtered buffer

Gel picture

insert jpeg!!!
  • looks like there are residual staples in solution and the leading band is slower as the scaffold. Marty said this is normal, when purified samples are put on a gel.
  • new purification with less initial concentration and folding buffer as washing buffer
  • look at samples in the TEM as soon as possible!!!

Folded sample BM_1_1565 (23.8.2011) in Amicon (100 kDa) with 1 x FOBXM + 20 mM MgCl2

Sample filtering

  • 50 µl sample in a Amicon filter (100 kDa)
  • 400 µl 1xFOBXM20 added
  • spinned for 5 minutes at 14000 rcf
  • 400 µl 1xFOBXM20 added
  • spinned for 5 minutes at 14000 rcf
  • 400 µl 1xFOBXM20 added
  • spinned for 5 minutes at 14000 rcf
  • turned the filter and sinned the purified sample into a new Epi for 2 minutes at 1000 rcf
  • 1 x and 2 x filtered buffer filled into separate Epi (the buffer, which came through the filter; if purification with spin columns works, this should only contain staples)
  • 3 x timed filtered buffer discarded

Ran a 125 ml 2% Agarose-Gel with EtBr

Photometric spectroscopy of MH255_647 and MH256_550 adding ErBr

To compare results with RTPCR

sample preparation

preliminary considerations

  • DNA with and without labels
  • with Label: 2x50µl
  • without label 1x50µl
  • Target Concentrations for EtBr: 0, 0.044, 0.4, 7.4 (in Units of Kd)

stocks

  • Buffer: 0.5 TE + 11mM MgCl2
  • 200 µM EtBr: 1.58µl EtBr (25mM) + 198.4µl Buffer
  • 50 µM EtBr: 25µl EtBr (200µM) + 75µl Buffer
  • 280nM MH255 647N
  • 710nM MH256 550

samples

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