Week 5

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  • There are no colonies for C0012 , so we inoculate I52034 and B0015 colony in 5 ml.
  • We strake on plates 30/07 ligations.
  • We get along with the J04431 fluorescence test:

-From the glycerol stock we sprout 1 ml at 37°C for 1 hour in agitation.

-Every 15 minutes we check bacterial OD and fluorescence. Cells are fluorescent at t= 210 min and OD=0.5.

  • We decide to repeat the test to understand if we can switch off the fluorescent cells with a higher amount of glucose.

We will perform this test on 07/08.

  • We get along with:

-Miniprep of I52034 and B0015;

-Digestion of B0015 with Xba/Pst1;

-Digestion of I52034 with Spe/Pst1;

- Gel extraction (we can’t see B0015);

-R0015+I13507(I763007) ligation which doesn’t give colonies so we transform with the other 10 ul of ligation;

-J04500+C0051(I763005)ligation which gives colonies so we inoculate in 5ml;

-Transformation for C0012 (4ul) and for J22101 (2ul);

-A new R0051+ I13507 (I763007) (vector 4ul, insert 8ul) ligation.

  • Today we do:

-Miniprep of J04500+C0051 (I763005);

-Control digestion of 5 ul with Eco/Pst1;

-A new B0015 digestion with Xba/Pst1;

-Run on electroforesis gel. We have problems with gel because we don’t see the band;

-Band extraction for B0015 e I763005;

-There are no colonies for C0012, but there are colonies for R0051 + I13507(I763007). So, we identify the correct ligation protocol;

- R0010, P0412, C0012 transformation;

-Inoculation in 5 ml for R0051 + I13507 and for J22101;

- R0051 + I13507 (I763007) fluorescence test to check if the RFP filter works properly.

  • We get along with:

-Miniprep for J22101 and for R0051+ I13507 (I763007);

- J22101 digestion with Xba/Pst1 ;

- Control digestion of R0051 + I13507 (I763007);

- J04500+C0051 (I763005) and B0015and J22101 and R0051 + I13507 (I763007) run on electroforesis gel and J04500+C0051 (I763005) and J22101 extraction;

-Glycerol stocks preparation;