Team:Heidelberg/Notebook/Sensing Group/Notebook/2ndweek

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Monday, 08/11/2008

Miniprep of correctly sequenced LuxS and Glycerol-stock
LuxQ no. 3 checked via digestion with NcoI/BamHI (NEBuffer 3 + BSA) --> negative result
PCR for LuxQ, LuxQ-1, LuxQ-2, Tar-1, Tar-2 with Taq Mastermix
- 5min @ 95°C || 30s @ 95°C | 30s @ 58 °C | 2min @ 72°C || 10min @ 72°C | 4°C hold (30 cycles)
preparation of O/N culture for LuxQ no. 3

PCR-purification of the all PCR-products eluted in 50 µl H₂O
Miniprep of LuxQ no. 3 O/N culture and digestion with xbaI (NEBuffer 2 + BSA)
Fusion-PCR for Fusion-1 and repetition of PCR for LuxQ-2 and LuxQ
digestion of pDK48 plasmid and Fusion-1 frament with NcoI/NdeI (0.5 µl) and AgeI (1 µl) in NEBuffer 4

Fusion-PCR for Fusion-1, LuxQ PCR, LuxQ-2 PCR, pDK48 miniprep and xbaI digestion of LuxQ no. 3

Digestion of Fusion-1 and pDK48 with NcoI/NdeI/AgeI

PCR for LuxQ and LuxQ-2

digestion of Fusion-1 with AgeI in NEBuffer 1
gel extraction of sequentially digested Fusion-1 and pDK48 products
new digestion of Fusion-1 with NcoI/NdeI (NEBuffer 4) and gel extraction, because previous digestion did not yield expected bands (there should be two small bands at 516bp and 392bp)

Gelextraction of digested Fusion-1 and purified PCR product as well as pDK48 digested. Expected bands at 516bp and 392bp are not visible.

Fusion-1 digested with NcoI/NdeI. Expected bands of 1025bp and 908bp had to be cut out together. Wanted band is the longer one.