Team:Heidelberg/Templates/M-04-05-13

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Revision as of 00:08, 4 October 2013

Contents

Cloning

  • DelH (review of the work done so far)
    • primers were designed and ordered
    • DelH gene first try with plamsid backbone: pSB6A1
      • AraC Promoter, RBs, lacZ, low ori pMB1, amp-R
      • PacI and KpnI are not in DelH gene, thus used for ligation
    • DelA to P into single plasmid with about 26kbp: pSB4K5
      • lacI Promoter, RBs, mRFP1, low ori pSC101, Kan-R
    • Hanna's work so far: Top 10 E.coli
      • pSB4K5 with Insert
      • pSB6A1
      • AraC
      • lacZ
      • pSB1C3 with insert 
  Are supposed to be joined by cloning to complete backbones by Monday
  • Indigoidine
    • similar to DelH
      • pSB1C3
      • high ori 0034, lacI promoter, mRFP1, Cm-R
  • cloning with +TypeII RE (recognize sequence and then cut at site one nucleotide next to recognition site)
    • division into three fragments

ask Dominik:

  • how exactly does this strategy work?
  • when is this an appropriate strategy to use?
  • Is "PCR Ligation" also a good Method (seperate PCRs for fragments; primers have "overlapping tail" takend from next fragment; ligation of fragments in one single PCR")?

Important! What about Freiburg? are they willing to help us with Gibson Cloning?

Approach of the Next Few Weeks

What should be bought: ---> Konrad's list

  • divison into smaller groups dealing with the main topics of our projects:
  1. Indigoidine (closed): Rald, Konrad, Nikos
  2. Del H lab protocol/methods (closed) : Hanna, Fanny, Sophie
  3. Del Rest (A to P) (closed) : Flo, Ilia, Nikos, Nils
  4. NRPS : Tania, Joschua, Ilia
  5. Software : Nils, Ilia, Konrad, Joschua, Hanna

Decisions Made

  • serial cloner should be our main software used for primer design etc.
  • Should use tool for oligo 2ndary structre prediction
    • http/mfold.rna.albany.edu
  • Should create common primer list; structure as in the following:
    • continous number, primer name, abbreviation (initials), sequence, details (if possible with a link to protocol used), creator, date, evaluation (e.g. worked, low yield ... )

Agenda for next meeting (13.05.2013 6 pm)

  • all groups meet up seperately and continue working
    • brief presentation on next meeting
  • next meeting (with Dominik)
    • progress report of individual groups and lab
    • prepping for upcoming big meeting
  • Next big meeting will be 15.5.2013
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