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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
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Latest revision as of 10:12, 18 September 2011