Week 12

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Revision as of 08:43, 3 October 2007 by Guido Costa (Talk | contribs)
  • Ligations for:

-I763026 + I763019;

-I763026 + I763004;

-I763025 + J04031.

  • We transform ligations and strake them on plates.

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

  • Miniprep for:



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

  • Digestion for:

-I763028 with Eco/Spe;

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

-Ptet-LacI-GFP with Eco/Spe;

-I763007 with Eco/Xba;

-Plac-cI-LacY with Eco/Spe1;

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

-I763028, Ptet-LacI-T-PLac-GFP are died;

-Ptet-LacI-GFP is correct;

-I763007 not found;

-Plac-CI-LacY is correct.


Testing our devices

  • In 2 different tubes we add 5ml of LB medium and 2 different colonies of 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 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.