How to use the Notebook

template of a day

Calendar

12-07-2010

   * OD measurement of Asaia’s culture
   * Chemical competence for E.Coli DB3.1 (step : Day 3)
   * PCR (Asaia ORI + primers)
   * Made GLY agar plates without antibiotics -> failed (medium was too old)
   * Asaia O/N culture without antibiotics in order to recover some WT
   * Text for the sponsor 

13-07-2010

   * Chemical competence for E.Coli DB3.1 (step : Day 4) -> stored at -80°C
   * Preparation of GLY Medium / LB Medium / SOC Medium / TAE 1X / TAE 0.5X
   * Run a gel for the PCR -> failed (mix of two different kits)
   * Preparation of plates (GLY Agar / LB+Kan Agar / LB+Amp Agar)
   * Learned to autoclave
   * Restarted PCR (Asaia ORI + primers)
   * Plated Asaia (O/N culture)
   * Choice of Antibiotics Biobricks resistance (Kan / Amp / Tet / Chl) from the iGem Kit and preparation of them (streak out)
   * Transformation of E.coli DB3.1 with pUC19 to check its competence
   * Transformation of E.coli DH5 with p1003 (Kan) / p1002 (Amp) / p1004 (Chl) / p1005 (Tet)
   * E.coli DB3.1 and DH5 were plated on LB+Amp Agar plates (Kan was plated on LB+Kan Agar plate)
   * Ordered material
   * Wiki brainstorming
   * Protocols printing and organization 

14-07-2010

   * Run a gel for the PCR of the previous day -> worked
   * Purification of PCR’s product -> ok
   * Preparation of the BBa_151020 (streak out)
   * Competence Calculation of E.Coli DB3.1 (+pUC10, Amp) -> ok
   * Look at Asaia with microscope -> we have cells (pictures)
   * Growth Curve for Asaia (OD measurement each hour) -> both manually (ok) + with the plate reader
   * Plated Asaia to recover some WT (streak out)
   * Preparation of Asaia Culture (for Lemaître experiment) with Kan
   * Protocol LateX template
   * O/N culture of the E.coli DH5 transformed with the BB Antibiotics resistance (4x) -> miniprep
   * Learning HTML for the wiki
   * Text for the project presentation due to iGem
   * Writing Protocols of basic procedures for the wiki page
   * Culture of Asaia + Kan 

15-07-2010

   * MaxiPrep of Lemaître’s culture
   * Purification of the BB resistances from the E.Coli cultures
   * Protocol for cloning the Asaia Vector
   * Enzyme digestion of the BB resistances and Asaia_ORI
   * Cloning (Asaia_ORI + Vector/ Asaia_ORI + BB + Vector)
   * Wiki text
   * Preparation of LB agar + Cm
   * Electrophoresis of resistance fragments (Works!!!) and extraction
   * Purification of extracted DNA from agarose gel 

16-07-2010

   * ligation Asaia origin BB + vector
   * ligation Asaia origin BB + Kan + vector
   * ligation Asaia origin BB + Amp + vector
   * ligation Asaia origin BB + Cm + vector
   * ligation Asaia origin BB + Tet + vector
   * Extraction of the OD curve data (values)
   * Replating Asaia on plates with and without kanamicin in order to recover the WT Asaia (only WT do not grow on kanamicin)
   * Feeding and infection of Drosophila (WT and immunodeficient redish mutants) with bacteria Ecc15 and Asaia in Prof. Lemaitre Lab
   * Fluorescence microscopy -> presence of fluorescence in the body/gut as expected
   * Transformation of E-Coli with the ligation products and plating the transformed bacteria 

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 

26-07-2010

   * Lemaître experiment: 400 ml cultures of GFP Asaia, Ecc15 and P.Entomophila
   * Lemaître experiment: separating male and female drosophila
   * Transformation of the Base Vector with -Kan-ori- insert into E.Coli DB3.1
   * PCR of LovTab as a positive control to check if the PCR works (with own and Henrike's dNTP's) --> Run gel
   * Test-digestion of some of the Mini-preps (Ori alone, Ori-Amp) --> Run gel
   * Recovered the wildtype Asaia worked!!
   * WT Asaia strains arrived. Culture started 

27-07-2010

   *  Ligation C3/A3 + Ori + resistance (Kan, Amp, Tet, Cm)
   * transformation of the ligation products into E.Coli DH5a
   * Plating of transformed cells with selection of the vector and the insert
   * Colony PCR of E.Coli DB3.1 containing BaseVector + Ori + Kan --> running gel (twice, the first time failed)
   * Preculture of (maybe!!) WT Asaia for competence protocol 
   * Sorting of male/female flies
   * Culture of 400 ml of different species of bacteria. 

28-07-2010

   * Analysis of plating of transformed cells
   * Gel for check the Colony PCR
   * 250 ml Gly medium
   * OD measurement of preculture for competent Asaia --> too high. Start a new one!!!
   * Competent Asaia and stocking @-80°C
   * Plating of transformed E.Coli for double check 
   * preparation of the pellets for infection with 3 different species of bacteria + OD measurements
   * preparation of tubes with 15 female flies
   * begin of the survival experiment: infection of flies --> check of the flies after 2h 

29-07-2010

   * Results from plating of transformed E.Coli for double check and miniprep from some of them
   * Digestion with different restriction enzymes to check if we get the right length
   * Counting of dead flies one day after the infection
   * Observation of fluorescence of infected flies under the microscope 

30-07-2010

   * Running a gel of the digested products of miniprep (29.07) --> bad results
   * Miniprep for the last 17 samples of replating
   * Digestion of 5 samples with 3 restriction enzymes to check if we get the fragments we expect
   * Running a gel for the digestion products
   * Counting of dead flies two days after the infection
   * Observation of fluorescence of infected flies under the microscope 

02-08-2010