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
- 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
- all five flow through bufer filled into separate Epi (the buffer, which came through the filter; if purification with spin columns works, this should only contain staples)
Ran a 125 ml 2% Agarose-Gel with EtBr
- 1kb ladder
- p7560
- BM_1_1565 5xfiltered
- BM_1_1565 unfiltered
- 1x flow through
- 2x flow through
- 3x flow through
- 4x flow through
- 5x flow through
Gel picture
insert gel picture
Conclusion
Photometric spectroscopy of MH255_647 and MH256_550 adding EtBr
To compare results with RT-PCR
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
- incubate for 1min at 25°C
- incubate for 1min at 20°C
|
Project name
