Team:Heidelberg/Notebook/Sensing Group/Notebook/6thweek

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Monday, 09/08/2008

Liquid Culture of 5 colonies from LuxQ Transformation and 1 from F1 transformation
Miniprep of Liquid Culture from previous Transformation (done by Chenchen). Incubated for two days @ RT and about 6 h @ 37 °C
Digestion of the Miniprep products with xbaI (Buffer2 + BSA, 1 ul enzyme @ 37 °C about 40min)

Digestion of LuxQ with xbaI yielded expected bands (4736 + 1204 bp) for all samples, except for no. 4

Sequencing @ GATC yielded correct sequence for LuxQ no. 1

PCR for LuxQ and LuxP with Phusion (25 µl Mastermix, 0.5 µl each Primer, 0(1) µl DMSO, 24(23) µl H₂0, 0.5 µl Template)
PCR Programme: 5 min @ 98 °C || 10 s @ 98 °C | 30 s @ 55 °C | 30 s @ 72 °C || 5 min @ 72 °C | 4 °C

PCR for LuxQ and LuxP. LuxQ PCR did not work, but first PCR for LuxP was positive.

PCR for LuxQ fragment 1 and 2, Tar fragment 1 and 2 for Phusion receptor with Phusion-Polymerase
PCR Programme: 30 s @ 98 °C || 10 s @ 98 °C | 30 s @ 55 °C |30 s @ 72 °C || 5 min @ 72 °C | 4 °C (30 cycles)

PCR of LuxQ and Tar constructs for fusion receptor. LuxQ is negative

PCR of LuxQ constructs for fusion

Fusion-PCR