"
Team:EPF Lausanne/SecondWeek
From 2010.igem.org
(Difference between revisions)
| (11 intermediate revisions not shown) | |||
| Line 11: | Line 11: | ||
*Autoclaving of media and flasks | *Autoclaving of media and flasks | ||
| - | <h3>Tuesday | + | <h3>Tuesday 20.07.2010 </h3> |
| + | *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 | ||
| + | |||
| + | <h3>Wednesday 21.07.2010 </h3> | ||
| + | *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) | ||
| + | |||
| + | <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 | ||
Latest revision as of 17:37, 23 July 2010
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
