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

From OpenWetWare
Jump to navigationJump to search
Line 6: Line 6:
| colspan="2"|
| colspan="2"|
<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### -->
<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### -->
=[[Size exclusion filter purification]]=
=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]]==
==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]]===
===Sample filtering===
* 50 µl from both samples were mixed and put into a Amicon filter (100 kDa)
* 50 µl from both samples were mixed and put into a Amicon filter (100 kDa)
* 400 µl 0.5x TBE + 11mM MgCl2 added
* 400 µl 0.5x TBE + 11mM MgCl2 added
Line 22: Line 22:
* 3 x timed filtered buffer discarded
* 3 x timed filtered buffer discarded


===[[Ran a 50 ml 2% Agarose-Gel with EtBr]]===
===Ran a 50 ml 2% Agarose-Gel with EtBr===




Line 33: Line 33:
* 2x filtered buffer
* 2x filtered buffer


===[[Gel picture]]===
===Gel picture===


  insert jpeg!!!
  insert jpeg!!!
Line 41: Line 41:
* look at samples in the TEM as soon as possible!!!
* 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]]==
==Folded sample BM_1_1565 (23.8.2011) in Amicon (100 kDa) with 1 x FOBXM + 20 mM MgCl2==


===[[Sample filtering]]===
===Sample filtering===


* 50 µl sample in a Amicon filter (100 kDa)
* 50 µl sample in a Amicon filter (100 kDa)
Line 60: Line 60:
* 3 x timed filtered buffer discarded
* 3 x timed filtered buffer discarded


===[[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=
=Photometric spectroscopy of MH255_647 and MH256_550 adding ErBr=

Revision as of 06:13, 6 September 2011

Project name <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>

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
  • 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

Labled DNA

concentration EtBr (in Units of KD) EtBr Stock [µL] MH255_647N 280nM [µL] MH256_550 710nM [µL] buffer (0,5 TBE, 11mM MgCl2)
0 0 3,6 1,4 95
0.044 1,07 (50µM stock) 3,6 1,4 93,93
0.4 9,6 (50µM stock) 3,6 1,4 85,4
7.6 45.6 (200µM stock) 3,6 1,4 49,4


Control: Unlabled DNA

concentration EtBr (in Units of KD) EtBr Stock [µL] MH255 280nM [µL] MH256 710nM [µL] buffer (0,5 TBE, 11mM MgCl2)
0 0 0.5 0.5 49
0.044 0.54 (50µM stock) 0.5 0.5 48.47
0.4 4.8 (50µM stock) 0.5 0.5 44.2
7.6 22.8 (200µM stock) 0.5 0.5 26.2

Hybidisation:

Incubation for 2min at 65°C, decrease temperature 1°C/min



Retrieved from "https://openwetware.org/mediawiki/index.php?title=Biomod/2011/TUM/TNT/LabbookA/2011/09/06&oldid=534392"