Team:EPF Lausanne/SecondWeek

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*Digestion of BB's produced and of the Base vector
*Digestion of BB's produced and of the Base vector
*Agar plates with Kan/Tet/Cm/Amp made
*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)  
*Prepare Tet stock (from powder)  
<h3>Thursday 22.07.2010 </h3>
<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>
<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

Latest revision as of 17:37, 23 July 2010

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Contents

Monday 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

Tuesday 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

Wednesday 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)

Thursday 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!!)

Friday 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