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

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Contents

Monday, 08/04/2008

Preparations

Preparations were started at the end of the week before

  • Transformation of pDK48 and pTrc99a in E. coli DH5a (0.5 µl plasmid-DNA + 50 µl competent cells)
  • Glycerol-stock of Vibrio harveyi BB120, BB886, mm30, BB178, BB125
  • picked pTrc99a and pDK48 cultures and inoculated in 3 ml LB overnight
  • V. harveyi cultured on LB+-Agar plate
  • MiniPrep of pTrc99a and pDK48 from DHha

Tuesday, 08/05/2008

Cloning of LuxP

LuxP colony PCR
LuxQ, LuxS PCR
  • PCR of LuxP from V. harveyi colony
  • 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 + 7 µl H2O
  • Ligation with vector:insert ratio 1:3 and 1:1

Cloning of LuxS

  • PCR of V. harveyi genome and PCR purification
  • digestion of LuxS and pTrc99a with BamHI/NcoI
  • Ligation with vector:insert ratio 1:3 and 1:1


Wednesday, 08/06/2008

  • 5 colonies of LuxQ,S,P transformation (1:1 ligation) picket and used for colony-PCR
  • Colony-PCR to check for insert. 5min @ 94°C || 30s @ 94°C | 30s @ 58 °C | 2min @ 72°C || 10min @ 72°C | 4°C hold (30 cycles)
Colony PCR of LuxQ, LuxS and LuxP
  • preparation of O/N cultures for Miniprep

Thursday, 08/07/2008

  • Miniprep of samples Q#1-5, S#1-5, P#1-5
  • PCR from Minipreps to confirm inserts
PCR to confirm insert from Minipreps. LuxQ no. 3 seems to be posivite. For LuxS and LuxP all samples are positive.
  • 10 new colonies of LuxQ-plate picked and checked via colony-PCR
LuxQ colony PCR of 10 new colonies.
  • ordered Sequencing of LuxS,P,Q positive clones at GATC 
Sequencing Results
LuxS: correct sequence
LuxP: all clones contain mutations, cloning has to be repeated
LuxQ: no insert in the picked colonies

Friday, 08/08/2008

  • Miniprep of overnight-cultures of the picked colonies Q#6-15
  • In silico cloning of LuxQ, S in pTrc99a and LuxP in pDK48
  • Test-Digestion of LuxQ with XbaI --> positive result for Q3, 7, 9, 11, 14


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