Team:EPF Lausanne/Notebook/week2

From 2010.igem.org

(Difference between revisions)
Line 42: Line 42:
-
[begin here]
+
=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

Revision as of 14:58, 1 September 2010









Contents

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



Retrieved from "http://2010.igem.org/Team:EPF_Lausanne/Notebook/week2"