IGEM:metu/2009/Notebook/wound dressing/2009/08/13

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13.08.2009

1. The transformed plates from the former day weren't successful. We couldn't observe colonies in many plates. It is appeared that the competent cells prepared at the last weekend are useless and we discarded them from the storage. Moreover, some plates are damaged because of shaking beads very much.

2. Digestion of Backbone 11, LuxR-RBS(11.08.2009) and pLacI(06.08.2009) are done.

 * Backbone 11 is cut with EcorI and PstI restriction enzymes.The following amounts are added to microtubes;
    1) 5.2 microliter plasmid
    2) 1 microliter EcorI
    3) 1 microliter PstI
    4) 4 microliter Buffer
    5) 28.8 microliter deionized water
 * LuxR-RBS(11.08.2009) is cut with XbaI and PstI restriction enzymes.The following amounts are added to microtubes;
    1) 2.65 microliter plasmid
    2) 1 microliter XbaI
    3) 1 microliter PstI
    4) 4 microliter Buffer
    5) 31.25 microliter deionized water
 * pLacI(06.08.2009) is cut with EcorI and SpeI restriction enzymes.The following amounts are added to microtubes;
    1) 2.65 microliter plasmid
    2) 1 microliter EcorI
    3) 1 microliter SpeI
    4) 4 microliter Buffer
    5) 31.3 microliter deionized water

The digestions of pLacI and Backbone were successful. However, LuxR-RBS isn't cut properly. We extracted the backbone and pLacI, and decided to store them at -20 C until LuxR-RBS is properly cut.

3. Transformation from the plates have been done. The plasmid to be transformated have been taken from 2009 plate 3 20K and 20M( ANOTHER LYSIS PLASMIDS). Transformation have been done on both the competent cells prepared at the morning and 2 weeks ago. Moreover, because of contaminated plates, the transformation of backbone and EGF series 4 have been proceeded.

4. The double digestion for LuxR-RBS has been proceeded with fastdigest enzymes again. Moreover, the plasmids of LuxR-RBS from 05.08.2009 has been also added. The control group (not digested LuxR-RBS) has been also added to the electrophoresis gel. The following amounts are added;

 * LuxR-RBS(11.08.2009) is cut with XbaI and PstI restriction enzymes.The following amounts are added to microtubes;
    1) 2.65 microliter plasmid
    2) 1 microliter XbaI
    3) 1 microliter PstI
    4) 4 microliter Buffer
    5) 31.35 microliter deionized water
 * LuxR-RBS(05.08.2009) is cut with XbaI and PstI restriction enzymes.The following amounts are added to microtubes;
    1) 2.71 microliter plasmid
    2) 1 microliter XbaI
    3) 1 microliter PstI
    4) 4 microliter Buffer
    5) 31.29 microliter deionized water


The results were not successful also. Double digestion didn't work. Our plasmids have been cut from only one side. It appeared that we should not perform double digestion on LuxR-RBS. The following day, the digestion of LuxR-RBS will be performed in two steps.