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Team:BIOTEC Dresden/Notebook2-2
From 2009.igem.org
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.
