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From 2013.igem.org

Contents 1 Cloning 2 Approach of the Next Few Weeks 3 Decisions Made 4 Agenda for next meeting (13.05.2013 6 pm)

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