Difference between revisions of "Team:Goettingen/Notebook"

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<a href="" onClick=" $('#menu14').slideToggle(300, function callback() {  }); return false;"><h2> 25.06.15</h2></a>
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    <div id="menu14">
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<p>
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    Present: Heiko, Ela, Julian, Dennis, Steffi, Avi, Verena, Alaa, Debbie, Nida
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</p>
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<p>
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    <strong>1.) Strain documentation: </strong>
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</p>
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<p>
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    The team has now got a box in the -80 ̊C freezer to start a strain collection. The Box is on the ground floor, in the middle freezer, top compartment, top
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    shelf marked: Rack 1 Robert at the back, 2<sup>nd</sup> from the bottom (blue box)
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</p>
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<p>
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    - To register the strain an information sheet needs to be filled in and submitted to Jaqueline
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</p>
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<p>
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    - Strains should be frozen from overnight culture in 30% glycerine (e.g. 600ul liquid culture + 900ul Glycerine (from 50% stock solution))
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</p>
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<p>
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    <strong>2.) Labwork:</strong>
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</p>
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<p>
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    Cellulase:
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</p>
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<p>
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    pJET_cellulase transformation and restriction worked fine in TOP10 and was then transformed into BL21 cells. Restriction showed a fragment of the correct
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    size, however sequencing failed. Activity plates showed no activity so far, but will be incubated over a longer period of time and in addition singularised
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    to avoid contamination of other colonies.
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</p>
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<p>
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    Scaffoldin:
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</p>
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<p>
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    Trafo in Top10 and BL21 did not work, which is why the TOPO cloning may be problematic.
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</p>
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<p>
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    - A control with an empty vector should be run.
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</p>
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<p>
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    - If the annealing is successful the bands should change in a reaction, which can be verified to exclude those issues.
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</p>
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<p>
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    - In addition Primers should be designed from within the sequence to verify the ends of the scaffoldin. Primers should be around 18bp long and 200bp within
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    the construct, which also has TOPO overlaps
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</p>
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<p>
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    - In addition the scaffoldin band of the PCR could be correct, but with an impurity alongside -&gt; transformation should however still work to some extent
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    (second problem?)
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</p>
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<p>
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    - For the covalent binding stick to the protocol an do not reduce salt concentrations
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</p>
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<p>
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    - For electroporation the TOPO cloning mixture needs to be desalinated with microdialysis, otherwise the trafo won't work
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</p>
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<p>
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    Recommendation: Check salts, positive control, annealing conditions and sequencing after PCR purification.
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</p>
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<p>
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    RFP:
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</p>
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<p>
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    Sequences are 100%match, but colonies show no fluorescence. Therefore, restricted RFP sequences will be ligated into pBAD in order to verify its function
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    in the target plasmid.
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</p>
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<p>
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    GFP sequences for Primer design and corresponding plasmids/colonies can be acquired from Jaqueline.
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</p>
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<p>
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    E1&amp;2:
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</p>
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<p>
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    Sequencing and restriction control were ok. Strains are frozen and documentation is being prepared.
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</p>
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<p>
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    Ask Silja for origin of the enzymes for better documentation. One is from Heiko Nacke, the other from Genis. In addition the enzyme sequences can be
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    Blasted for identification.
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</p>
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<p>
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    Phosphatase contains PstI, which is why currently it is not compatible with biobricks. In the future the restriction sites will have to be changed, if
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    submission should occur (PCR modification).
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</p>
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<p>
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    Dockerins:
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</p>
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<p>
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    Heat shock transformation did not work for any of the 4 dockerins, but positive control worked fine.
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</p>
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<p>
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    Vector was cut with EcoRI and KpnI as a test and KpnI was not very efficient. Due to this the restrictions were done separately.
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</p>
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<p>
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    Next step is a ligation and a gel to verify it immediately after to find reasons for failure:
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</p>
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<p>
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    - T4 ligase working?
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</p>
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<p>
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    - Phosphorylation issues -&gt; skip this step
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</p>
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<p>
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    - Find Plasmid formula to calculate ideal ligation ratio (depends on insert length and is usually 3:1) -&gt; overloading the vector may cause different
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    ligation variants
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</p>
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<p>
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    - Can E.coli express the dockerins?
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</p>
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<p>
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    Oder dockerins as codon optimised gBlocks from IDT, as a backup.
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</p>
 
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Revision as of 19:37, 12 September 2015



Notebook

15.12.14

08.01.15

13.01.15

15.01.15

19.01.15

02.02.15

16.02.15

18.02.15

23.02.15

16.03.15

23.3.15

07.04.15

19.0615

25.06.15