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AUGUST: WEEK 5
August, 29th
T9002-ENTERo and ENTERO-RBS were diluted 1:500 in M9 + Amp + Cm12.5 13 ml and 1 ml volume respectively. After six hour growth they were aliquoted in 96-well microplate (190 μl cultures) with 10 μl of supernatants collected on August, 27th and 10 μl of 3OC6-HSL.
Plates incubated on August, 27th were all grown, except for J101-31-1C3; for each of them a colony was picked and inoculated in LB + Cm34, 5 ml, while for J101-E7 two colonies were inoculated in LB + Cm34, 5 ml.
August, 30th
Glycerol stocks were prepared for E2-1C3, E3-1C3, E4-1C3, E5-1C3, E6-1C3, E7-1C3, E9-1C3, E10-1C3, E11-1C3, J101-E5-1C3, J101-E7-1C3-1 and J101-e7-1C3-2.
Each sample was digested with EcoRI and PstI endonucleases (a mix 13 x was prepared for high copy number plasmid parts):
while for E43-2 10 μl were digested. In the afternoon the parts were screened by gel electrophoresis:
All parts showed the correct insert length except for E3-1C3. We decided to pick and inoculate another colony from the plate, named E3-1C3-2.
August, 31st
E2-1C3, E4-1C3, E5-1C3, E6-1C3, E7-1C3, E9-1C3, E10-1C3, E11-1C3, J101-E5-1C3, J101-E7-1C3-1, E43-2 and E43-3 DNA samples were prepared and sent to BMR Genomics for sequencing.
These plasmids were digested with EcoRI and PstI endonucleases to transfer them in pSB1C3:
In gel electrophoresis all inserts showed the correct length: After gel-extraction digested DNA was quantified:
Then ligations were performed in a final volume of 10 μl:
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Team:UNIPV-Pavia/Calendar/August/week5
From 2011.igem.org
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- | All parts showed the correct insert length except for E3-1C3. | + | All parts showed the correct insert length except for E3-1C3. We decided to pick and inoculate another colony from the plate, named E3-1C3-2. |
<br> | <br> | ||
40 μl CaCl<sub><small>2</small></sub> 1M, 800 μl Casamino Acids 10 %, 80 μl MgSO<sub><small>4</small></sub> 1M and 1.36 ml thiamine were added to previously autoclaved flasks to prepare M9 at different pHs. | 40 μl CaCl<sub><small>2</small></sub> 1M, 800 μl Casamino Acids 10 %, 80 μl MgSO<sub><small>4</small></sub> 1M and 1.36 ml thiamine were added to previously autoclaved flasks to prepare M9 at different pHs. | ||
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E2-1C3, E4-1C3, E5-1C3, E6-1C3, E7-1C3, E9-1C3, E10-1C3, E11-1C3, J101-E5-1C3, J101-E7-1C3-1, E43-2 and E43-3 DNA samples were prepared and sent to BMR Genomics for sequencing. | E2-1C3, E4-1C3, E5-1C3, E6-1C3, E7-1C3, E9-1C3, E10-1C3, E11-1C3, J101-E5-1C3, J101-E7-1C3-1, E43-2 and E43-3 DNA samples were prepared and sent to BMR Genomics for sequencing. | ||
<br> | <br> | ||
- | ENTERO-4C5 cutlures were diulted 1:200 in a final volume of 5 ml M9. After 2 hours 3OC<small><sub>6</sub></small>-HSL was added to a final | + | ENTERO-4C5 cutlures were diulted 1:200 in a final volume of 5 ml M9. After 2 hours 3OC<small><sub>6</sub></small>-HSL was added to a final concentration of 100 nM and the first supernatant sample was collected. After 1 hour, 4 hours and 22 hours from 3OC<small><sub>6</sub></small>-HSL supplementation samples were again collected; supernatants were all stored at -20°C in order to measure 3OC<small><sub>6</sub></small>-HSL concentration with <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> biosensor. |
<br> | <br> | ||
BBa_R0040 in J61002, E3-1 and J101-31 were extracted with MiniPrep kit; here are their quantifications: | BBa_R0040 in J61002, E3-1 and J101-31 were extracted with MiniPrep kit; here are their quantifications: | ||
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</table> | </table> | ||
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+ | Glycerol stock was prepared for E3-1C3-2 and the culture was pelletted. | ||
Revision as of 14:36, 2 September 2011