Team:BIOTEC Dresden/Notebook2-2

6th September’09:

1. A Red/ET recombination has been done to insert PCR product of Spec pR6K-Amp into GB05MN fosmid-BsdR and plated on LB-Cm10.Spec50 plates for overnight incubation.

8th September’09:

1. Inoculation of GB05FosmidBsdR in LB-Cm10 and GB05-pSC101RhaFlp-TetFlp RedαβγRecA has been done in LB-Amp100 and incubated at 37˚C and 30˚C respectively overnight.

9th September’09:

1. A repeat Red/ET recombination has been done to insert PCR product of Spec pR6K-Amp and into GB05MN fosmid-BsdR and plated on LB-Cm10.Spec50 plates for overnight incubation.

2. A Red/ET recombination has been done to insert PCR product of Spec pR6K-Amp and fosmid-BsdR into GB05MN pSC101RhaFlp-TetFlp RedαβγRecA fosmid and plated on LB-Cm10.Spec50 plates for overnight incubation.

3. Re-electroporation of pRhaFlp-CmR has been done for the positice clones.

10th September’09:

1. Reinoculation of colonies from the re-electroporated plates of pRhaFlp-CmR in LB-Amp liquid and solid agar plates.

2. A repeat Red/ET recombination has been done to insert PCR product of Spec pR6K-Amp and into GB05MN fosmid-BsdR and plated on LB-Cm10.Amp100 plates for overnight incubation.

3. A sequencing reaction has been set up for the positive clones of pTetFlp-KanR with 5’out and 3’out primers using Taq polymerase.

4. A Red/ET recombination has been done to insert PCR product of Kan2 into Red cells and plated on LB-Kan15 plates for overnight incubation.

11th September’09:

1. Minipreps of GB05 RhaFlp-CmR clone has been done and concentration is measured.