Team:BIOTEC Dresden/Notebook1-3

26th August:

1. A PCR reaction has been set up with pR6K-CmR and pR6K-KanR as templates with Kan-R/Kan-F and CmR/CmF primer pairs.

2. The PCR product has been run on a agarose gel to check quality and later the concentrations are also measured with Nanodrop.

3. Electroporation of plasmid pR6K-CmR has been done with Pirate competent cells and eventually incubated on LB-Cm15 plates to check for colonies.

4. Inoculatiuon of pRhaFlp number 6 has been done in LB-Amp and of ptetFlp in LB-Hyg and incubated overnight at 37 degrees shaking.

27th August:

1. Microfluidic templates have been prepared following a standard protocol.

2. A Red/ET recombination induction step has been carried out for the plasmid pRhaFlp with anhydrotetracycline anf pTetFlp with L-rhamnose and a electroporation has been done at 1350Volts, followed by recovery of cells and plating and incubating overnight for colonies.

28th August:

1. Colonies from pTetFlp-KanR plates are picked and re-inoculated in LB-Kan15 plates.

2. The GB05 strain is tested for growth in Trimethoprin LB-medium.

29th August:

1. OD measurements of wild type GB05cells and DhfR cells has been done by a spectrophotometer.

2. Minipreps of pTetFlp-kanR has been done.

31st August:

1. The concentrations of the minipreps of pTetFlp-KanR has been measured using nanodrop.

2. A restriction digest of the minipreps of pTetFlp-KanR and parental plasmid pRedFlp has been done with PstI.

3. The digests are run on a 1% agarose gel and the uncut dna on a 0.9% gel.