Biomod/2014/Scaffold: Difference between revisions

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<td> DNA origami scaffold Scaffold production The scaffold (8064 nucleotides) used for the DNA origami is the ssDNA of bacteriophage M13mp18, the staples were bought from MWG Eurofins Genomics. M13mp18 is a filamentous bacteriophage, which needs a host cell to reproduce. Therefore the Escherichia coli strain Top10F´ is infected with the phage. The Top10F´ bacteria cells are resistant to tetracycline and streptomycin so that a selection is possible. Because M13mp18 is a non-lytic phage, the phage infection is not lethal for the Top10F´ bacteria cells. But the infection is visible by plaques in E. coli caused by a decreased cell growth. Phage purification Before the ssDNA was extracted, the bacteriophage was purified to avoid undesirable mutations. At first an overnight culture of Top10F´ bacteria cells on a LB-Agar plate containing 20 µg/mL Tetracycline were prepared and incubated at 37°C. The next day 1 mL DYT medium was inoculated with one clone of Top10F´cells and shaken at 37°C for 6 h. Meanwhile the phages were diluted with DYT medium (dilution factors: 10-3, 10-5, 10-7, 10-9, 10-11) and soft agar was prepared out of 50 mL LB medium and 50 mL LB-agar medium which are heated to 50 – 60°C. 50 µL of the cultured Top10F´cells were infected with 100 µL of each phage dilution and added to 2.5 mL soft agar. After gently mixing the soft agar was put on ready-made agar plate and incubated at 37°C over night. Out of the plate with the 10-11 dilution phage three plaques were picked and incubated in 2 mL DYT medium each at 37°C and 120 rpm over night. The next steps were carried out for the three phage plaques respectively. An overnight culture of Top10F´cells in 2 mL DYT medium was also incubated at the same conditions. The next day the phage solution was centrifuged at 3000 g for 15 min, the supernatant was decanted and the centrifugation repeated twice. After incubation of 100 µL Top10F´cells of the overnight preculture in 10 mL DYT medium for 3 h at 37°C and 120 rpm these cells were infected with 100 µL phage solution (supernatant) and incubated over night at 37°C and 120 rpm. The rest of phage solution was stored at 4°C. The cell-phage solutions were centrifuged at 3000 g for 20 min. The supernatant was decanted and the centrifugation step repeated twice. (The pellet can be used for isolation of ds M13mp18 bacteriophage DNA). The supernatant is used for the ss M13mp18 bacteriophage DNA isolation. Preparation of bacteriophage M13mp18 ssDNA 50 mL of LB medium were inoculated with 100 µL Top10F´ bacteria cells and incubated at 37°C and 160 rpm overnight. The next morning 400 mL prewarmed (37°C) DYT-Medium with 5 mM MgCl2 were inoculated with 4 mL pregrown Top10F´ bacteria cells. The suspension was incubated at 37°C and 150 rpm till the OD reaches a value of 0.5 (cell density of 48 cells/mL). This suspension was infected with 200 µL phage solution (supernatant obtained by phage purification) and incubated for 5 h at 37°C and 150 rpm. The suspension was centrifuged at 3000 g and 4°C for 30 min, the supernatant was decanted and centrifuged again at the same conditions to separate the bacterial cells. The supernatant was stored over night at 4°C. For the bacteriophage precipitating 80 mL buffer M1 were added to the supernatant, mixed and incubated for 1 h at 4°C. The suspension was centrifuged at 10000 g and 4°C for 30 min. The supernatant was discarded. The pellet was resuspended in 7.5 mL 10 mM Tris pH 8.5 and incubated on ice for 1 h. For ssDNA extraction 7.5 mL of buffer PPB2 were added to the suspension, mixed by inverting and incubated on ice for 3 min. Then 5.25 mL buffer PPB3 were added and incubated on ice for 10 min after mixing. The suspension was centrifuged at 16500 g and 4°C for 30 min. The supernatant was decanted and centrifuged again at 16500 g and 4°C for 15 min to separate proteins. The ssDNA was precipitated by adding 0.1 volumes of PPB3 and 2 volumes of ethanol absolute. After mixing by inversion the suspension was incubated at -20°C for 40 min and centrifuged at 16500 g and 4°C for 60 min. The supernatant was carefully removed. The pellet was air-dried and dissolved in 1 mL 10 mM Tris ph 8.5. The concentration of ssDNA was adjusted to 100 nM with 10 mM Tris pH 8.5.

Buffer and solutions:

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<tr> <td align="left" valign="top"> LB medium:</td>

   <td><p align="justify"; style="line-height:2em"><font size="3pt">10 g/L NaCl<br>

10 g/L Bacto-tryptone 5 g/L Yeast extract in ddH2O, autoclave </td> </tr> <tr><td align="left" valign="top">DYT medium:</td>

   <td><p align="justify"; style="line-height:2em"><font size="3pt">16 g/L Bacto-tryptone<br>

10 g/L Yeast extract 5 g/L NaCl in ddH2O, autoclave </td> </tr> <tr><td align="left" valign="top">Buffer M1:</td>

   <td><p align="justify"; style="line-height:2em"><font size="3pt">150 g PEG-8000/500 mL<br>

87.66 g NaCl/500 mL </td> </tr> <tr><td align="left" valign="top">10mM Tris:</td>

   <td><p align="justify"; style="line-height:2em"><font size="3pt">121.14 mg Tris base /100 mL<br>

Adjust to pH 8.5 with HCl </td> </tr> <tr><td align="left" valign="top">Buffer PPB2:</td>

   <td><p align="justify"; style="line-height:2em"><font size="3pt">1.6 g NaOH/200 mL<br>2 g SDS/200 mL</td>

</tr> <tr><td align="left" valign="top">Buffer PPB3:</td>

   <td><p align="justify"; style="line-height:2em"><font size="3pt">58.88 g C<sub>2</sub>H<sub>3</sub>KO<sub>2</sub>/200 mL<br>Adjust pH to 5.5 with glacial acetic acid</td>

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Biomod/2014/Scaffold: Difference between revisions

Revision as of 11:03, 23 October 2014

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