Team:EPF Lausanne/SecondWeek

(Difference between revisions)

Latest revision as of 17:37, 23 July 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

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

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)

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!!)

@@ Line 30: / Line 30: @@
 *Digestion of BB's produced and of the Base vector
 *Agar plates with Kan/Tet/Cm/Amp made
-*Run Gel with the products of the digestion to check if the fragments are as expected
+*Prepared Gel with the products of the digestion to check if the fragments are as expected
 *Prepare Tet stock (from powder)
 <h3>Thursday 22.07.2010 </h3>
+*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!!)
 <h3>Friday 23.07.2010 </h3>
+*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