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

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Monday, 09/08/2008

Culture of 5 colonies from LuxQ Transformation and 1 from F1 transformation
Miniprep of Culture from previous Transformation (done by Chenchen). Incubated for two days @ RT and about then 6 h @ 37 °C
Digestion of the Miniprep products with XbaI (NEBuffer 2 + 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 verified 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

LuxP digestion with NcoI. All colonies are positive

Sequencing @ GATC: LuxP1 correct

PCR for LuxQ 1+2, Tar 1+2 and Gel Extraction to get rid of first primers
Fusion-PCR of LuxQ1+Tar1 und LuxQ2+Tar2 with Phusion and 55 °C annealing --> No product
Fusion PCR of LuxQ fragment 1b, LuxQ fragment 1c, LuxQ fragment 2b, LuxQ fragment 2c, Tar fragment 1b, Tar fragment 1c, Tar fragment 2b, Tar fragment 2c with Phusion and 55 °C annealing. Products were purified via Gelextraction to get rid of first primers
Two Fusion PCR (30 s @ 98 °C || 10 s @ 98 °C | 30 s @ 55 °C |30 s @ 72 °C || 5 min @ 72 °C | 4 °C (30 cycles)) with PCR purification.
Gel Extraction of LuxQ 1+2, Tar 1+2

First Fusion PCR

another fusion PCR

PCR Purification of previous Fusion PCR
Digestion with NcoI/NdeI (NEBuffer4) and NcoI/KpnI (NEBuffer1 + BSA). 1 h @ 37 °C
Gel extraction, eluted in 30 µl water
Ligation (Insert:Vector 5 µl:5 µl) and transformation into DH5alpha competent cells

nothing to report