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Team:Heidelberg/Templates/M-04-05-13
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== Cloning == | == Cloning == | ||
* '''DelH (review of the work done so far)''' | * '''DelH (review of the work done so far)''' | ||
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'''Important! What about Freiburg? are they willing to help us with Gibson Cloning?''' | '''Important! What about Freiburg? are they willing to help us with Gibson Cloning?''' | ||
| - | = Approach of the Next Few Weeks = | + | == Approach of the Next Few Weeks == |
What should be bought: | What should be bought: | ||
---> Konrad's list | ---> Konrad's list | ||
Revision as of 00:12, 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
- similar to DelH
- 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:
- Indigoidine (closed): Rald, Konrad, Nikos
- Del H lab protocol/methods (closed) : Hanna, Fanny, Sophie
- Del Rest (A to P) (closed) : Flo, Ilia, Nikos, Nils
- NRPS : Tania, Joschua, Ilia
- 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
