From 2007.igem.org

09/17/07

• Ligations for:

-[http://partsregistry.org/Part:BBa_I763026 I763026] + [http://partsregistry.org/Part:BBa_I763019 I763019];

-[http://partsregistry.org/Part:BBa_I763026 I763026] + [http://partsregistry.org/Part:BBa_I763004 I763004];

-[http://partsregistry.org/Part:BBa_I763025 I763025] + [http://partsregistry.org/Part:BBa_J04031 J04031].

• We transform ligations and strake them on plates.

09/18/07

• We inoculate a colony for yesterday ligations in 5ml of LB medium O/N.

09/19/07

• Miniprep for:

-[http://partsregistry.org/Part:BBa_I763028 I763028];

-Ptet-LacI-T-PLac-GFP;

-Ptet-LacI-GFP(Spe/Pst1), (Xba/Pst1).

• Digestion for:

-[http://partsregistry.org/Part:BBa_I763028 I763028] with Eco/Spe;

-Ptet-LacI-T-PLac-GFP with Eco/Spe;

-Ptet-LacI-GFP with Eco/Spe;

-[http://partsregistry.org/Part:BBa_I763007 I763007] with Eco/Xba;

-Plac-cI-LacY with Eco/Spe1;

• Band extraction from gel for all digestion and then we observe:

-[http://partsregistry.org/Part:BBa_I763028 I763028], Ptet-LacI-T-PLac-GFP are died;

-Ptet-LacI-GFP is correct;

-[http://partsregistry.org/Part:BBa_I763007 I763007] not found;

-Plac-CI-LacY is correct.

09/20/07

Testing our devices

• In 2 different tubes we add 5ml of LB medium and 2 different colonies of [http://partsregistry.org/Part:BBa_I763019 I763019] plasmid.
• In another tube we add 5ml of LB medium and a colony of PLac-cI-GFP plasmid.
• After 2 hours we measure the OD value and it is around 0.5.
• For each original tube we divide the bacteria fluid in two different tubes.
• We add the fluid without IPTG to one and the fluid with IPTG to the other one.

• The analyzed fluid without IPTG includes:

-2.5ml of original tube fluid,

-2.5ml of LB medium,

-2.5ul of kanamicin.

• The analyzed fluid with IPTG includes:

-2.5ml of original tube fluid,

-2.5ml of LB medium,

-2.5ul of kanamicin,

-50ul of IPTG.

• We examine a bacteria fluid drop from of each tube with our fluorescence microscopy.
• The bacteria with [http://partsregistry.org/Part:BBa_I763019 I763019] plasmid don’t beam fluorescence with and without IPTG;
• Very few bacteria with PLac-cI-GFP plasmid and without IPTG beam fluorescence;
• Several bacteria with PLac-cI-GFP plasmid with IPTG beam fluorescence.

• After 3 hours we measured the OD value of each tubes and it is 1.4.
• Bacteria are too concentrate, thus we dilute them.
• We take 2ml of LB medium, 1ml of bacteria fluid with and without IPTG and 2ul of kanamicin.
• Examining with fluorescence microscopy and we saw that only bacteria with PLac-cI-GFP plasmid beame fluorescence.

09/21/07

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