Team:Frankfurt/Notebook/Labwork

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= Labwork = __NOEDITSECTION__
Unfortunately making arrangements for the lab this year and applying for funding 
consumed more time than expected resulting in less time for the actual project.

== July 2013 == __NOEDITSECTION__
1. Arrangements for labwork such as preparing material <br>
* see protocols for the production of competent (''E.coli'', ''S.cerevisiae'') and corresponding media (LB, YEPD, SCD-ura,…)
2. Purchase/Organization of the equipment (reaction tubes, glass bottles, pipette tips,..)

== August 2013 == __NOEDITSECTION__
Recapitulation of the work done in 2012 : restriction digest of plasmids to make sure we can use them:
<br>
1. Digests with enzymes: BsaAI, EcoRI-HF, XbaI <br>
example:
* 1   µl DNA (~500nm)
* 0.5 µl BsaAI (5 units / µl -> one unit cuts once within one hour)
* 1   µl Buffer 4 (10x)
* 7,5 µl ddH2O
Expectation: 4 Bands with 2246, 3196, 2344, 3624 bp. Identification of one positive clone with the EcoRI-HF digest<br>
2. Production of competent DH5alpha <br>
3. Digest of plasmid p426 (mevalonate) with and without insert. <br>
4. Production of Cryo-Stocks of the p426 plasmids. <br>
5. Transformation with the positive clone <br>
6. Overnight cultures of yeast with the mevalonate plasmid. <br>
7. Making cultures of p426 with and without insert for GC usage. <br>
* Cultures were incubated for around 48 hours and harvested at an OD600 of 2.3 <br>
* Cells are centrifuged at 4000x g for 15 minutes.

== September 2013 == __NOEDITSECTION__
1. PCR of all fragments of the insert for the steviol plasmid (p423). <br>
* Since pHXT7 and tCYC1 are already on the plasmid p423 they do not have to be amplified. <br>
2. Making cultures for analysis with GC-MS <br>
3. Gel-Extraction of following fragments: CPS-KS, KO and KAH <br>
4. Production of comptent yeast cells (see protocols) <br>
5. Assembly of the Steviol plasmid with Gap-repair in yeast <br>

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