Week 11

From 2007.igem.org

(Difference between revisions)
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-pLac-cI-GFP with Xba/Pst1.
-pLac-cI-GFP with Xba/Pst1.
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*Band extraction from gel for all digestion except for pLac-cI-GFP. [[(photos)]]
+
*Band extraction from gel for all digestion except for pLac-cI-GFP. [[(photos2)]]

Revision as of 12:38, 20 September 2007

09/10/07
  • Miniprep for:

-I763027;

-I763028;

-I763019;

-I763021+P0412;

-I763021+S03520;

-I763021+S0100;

Digestion for:

-I763027 with Xba/Spe;

-I763028 with Spe/Pst1 and with Xba/Spe;

-I763019 with Xba/Spe;

-I763021+P0412 with Eco/Spe;

-I763021+S03520 with Eco/Spe;

-I763021+S0100 with Eco/Spe;

  • Band extraction from gel for all digestion;
  • We have problems with J52034 and with I763019.
  • Ligations for:

-I763028 + I763007

-S0100 + J04431, because we want to understand if LacI operates well;

-J06550 + J04631, with this ligation we want to understand if it operates well, because it doesn't leak.

09/11/07
  • Digestion for:

-I763020 with Xba/Pst1;

-J22101 with Xba/Pst1;


09/12/07
  • Control digestion before fluorescence tests. (photos)
  • Ligations for:

-I7633005(Spe/Pst1) + J04031(Xba/Pst1);

-S0100(Eco/Spe) + J04031(Eco/Xba);

-I763015 (Eco/Spe) + J04031(Eco/Xba).


09/13/07
  • We have find any colonies for I763015 + J04031;
  • Miniprep for:

- I7633005 + J04031;

-S0100 + J04031;

-I763028.

  • Control digestion for I763028.
  • M9 Medium and lactose stocks preparation.
  • Testing our devices

We take 5 ml of LB medium in which we put a colony of bacteria which contain Ptet-LacI Plac-cI-LacY-GFP plasmid. We examine a drop of it using our fluorescence microscopy setup. In these conditions, the bacteria don’t beam fluorescence. Starting from this moment, we put into the LB medium 1 mM of IPTG every 30 minutes until it is 4 mM , checking each time any eventual fluorescence, with negative results. After diluting 1 ml of the previous one with 4 ml of LB, we add IPTG until 1 ml of our new solution is 10 mM, then 20 mM and finally 40 mM. Despite that, no fluorescence is visible. For this reason now we test single parts of the plasmid. We take three populations of Plac-cI-LacY-GFP bacteria and one of Plac-cI-GFP bacteria. We add 1mM of IPTG to every population to check for fluorescence. We see the three Plac-cI-LacY-GFP populations produce a photomultiplier output between 5.55 and 5.61 Volts, while the Plac-cI-GFP population results more fluorescent giving a 5.91 Volts output. Every time we check for fluorescence, we use, as said, not only the photo camera but also the photomultiplier. Every measurement shows a 0.18 Volts offset due to environment light and a 1.30 Volts offset due to the excitation light at 501 nm. Since we know GFP gives a maximum of fluorescence when excited at 501 nm, we decide to go on with this wavelength despite the offset (a change of excitation wavelength reduces both the offset and the signal).



09/14/07
  • Glicerol stocks for:

-I763028;

-pLac-cI-GFP;

-I763020;

-I763026.

  • Fluorescence test for:

-I763028;

-pLac-cI-GFP;

-I763028(midi).

  • Miniprep for:

-I763028;

-pLac-cI-GFP;

-I763026;

-I763020.

  • Digestion for:

-I763028 with Eco/Xba;

-I763026 with Eco/Spe;

-S0100 with Spe/Pst1;

-S0100 with Eco/Spe;

-I763020 with Xba/Pst1;

-pLac-cI-GFP with Xba/Pst1.

  • Band extraction from gel for all digestion except for pLac-cI-GFP. (photos2)



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