Week 3

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