Team:Heidelberg/Notebook/Sensing Group/Notebook/1stweek

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Monday, 08/04/2008

Preparations were started at the end of the week before

Transformation of pDK48 and pTrc99alpha plamids in E. coli strain DH5alpha (0.5 µl plasmid-DNA + 50 µl competent cells)
Glycerol-stock of Vibrio harveyi BB120, BB886, mm30, BB178, BB125
pick pTrc99a and pDK48 colonies and then incubated in 3 ml LB at 37 overnight
V. harveyi cultured on LB plate
MiniPrep of pTrc99a and pDK48 from DH5alpha

LuxP colony PCR

LuxQ, LuxS PCR

PCR of LuxP from V. harveyi colony with the primers LuxPc and LuxPd with Taq polymerase Mastermix. 5min @ 95°C || 30s @ 95°C | 30s @ 58 °C | 2min @ 72°C || 10min @ 72°C | 4°C hold (30 cycles)
Gel Purification of LuxP eluted in 30 µl H₂O
Digestion of LuxP with SalI and NotI of pDK48 and LuxP-Gel-Purification product: 2 µl NEB-Buffer 3 + 0.5 µl SalI & NotI + 10 µl DNA + 6 µl H₂O
Ligation with vector:insert ratio 2µl:6µl and 5µl:5µl

PCR of V. harveyi genome with primers LuxSa and LuxSb and PCR purification 5min @ 95 °C || 30s @ 95°C | 30s @ 58 °C | 2min @ 72°C || 10min @ 72°C | 4°C hold (30 cycles) with Tag polymerase Mastermix
digestion of LuxS and pTrc99a with BamHI/NcoI (NEBuffer 3 + BSA)
Ligation with vector:insert ratio 2µl:6µl and 5µl:5µl

5 colonies of LuxQ,S,P transformation (1:1 ligation) picked and analyes with colony-PCR
Colony-PCR to check for insert. 5min @ 95°C || 30s @ 95°C | 30s @ 58 °C | 2min @ 72°C || 10min @ 72°C | 4°C hold (30 cycles)

Colony PCR of LuxQ, LuxS and LuxP

PCR to confirm insert from Minipreps. LuxQ no. 3 seems to be posivite. For LuxS and LuxP all samples are positive.

LuxQ colony PCR of 10 new colonies.

Sequencing Results
LuxS: correct sequence
LuxP: all clones contain mutations, cloning has to be repeated
LuxQ: no insert in the picked colonies