Week 2

19-07-2010

• Results of the transformation checked: successful!!
• Amplification of colonies (goal to do a miniprep of the plasmids --ori-- and --ori-resistance--)
• Results of plating of asaia culture (where we hope to recover the WT) checked: Asaia cultures grew on Kan plates also! Checked colonies for fluorescance under the microscope: no fluorescance --> Very unexpected! Don't know reason yet.
• Re-grow the colonies. Multiple cycles: hope that we get WT.
• Preparation of Asaia cultures for Lemaitre experiments/WT/Competence...
• Preparation of SOC medium and GLY medium
• Autoclaving of media and flasks

20-07-2010

• Plating Asaia to get WT (once again)
• Making HEPES Buffer for the Competence of Asaia
• Diluted the culture to get a 25ml culture(1:20) according to Asaia competence protocol
• OD curve plotted, Doubling time calculated
• No single colonies from the re-growing trial --> replating properly on separate plates
• Preparation of LB medium mixed with Kan/Amp/Cm and GLY medium mixed with <kan
• Miniprep of Origin + Resistance
• Miniprep of Origin alone.
• Miniprep of base vector BBa_151020
• Concentration measurments of miniprep products
• Glycerol stock of BB plasmid and Base Vector made

21-07-2010

• Measure OD of overnight culture of Asaia for competence --> value was too high
• Diluted culture to 0.3 OD, left in 30 degree incubator for 2 hours, OD of 0.6 measured --> start with competence
• Asaia competence
• Digestion of BB's produced and of the Base vector
• Agar plates with Kan/Tet/Cm/Amp made
• Prepared Gel with the products of the digestion to check if the fragments are as expected
• Prepare Tet stock (from powder)

22-07-2010

• Gel with products of digestion run --> only one of the base vector samples and a fragment with the origin and a Kan resistance worked. The others probably did not wok since the concentration of DNA was too low.
• Colonies picked and suspension cultures started. For making a miniprep tomorrow morning.
• Gel cut, Gel purification made
• New idea: PCR run on the product of the miniprep.
• Ligation of Base Vector with Origin+Kan
• Asaia preculture for OD growth (different pH) measurements
• Gly medium with pH 2,3,4,5,6,7.
• Research
• Replating of Asaia (Replating III) to get the WT (one day!!)

23-07-2010

• OD measurement growth curve in function of pH
• new DNA miniprep to extract Ori+AB_Res (the last one failed...)
• Lyse-n-Go protocol to do the ligase (and to check the insertion)
• transformation of the only digestion from yesterday that didn't failed (Kan)
• meeting with Onya for mosquito experiments
• preparation of 4L of Gly for LeMaitre experiments