Week 4

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  • We observe the uncorrected growth of I13507 on the plate and none of R0051.
  • We get along with:

- another transformation of R0051 (2 ul), I13507 and C0051;

- the ligation of J04500+J04631 (I763004) to test the ligation protocol with 2 experiences: vector and insert 2 ul, vector and insert 2-4 ul. We leave it at 25°C for an hour, then at 65°C for 10 minutes.

  • Then we transforme 4 ul of ligate.

  • We observe colony for C0051 on the plate. Then we preinoculate in 5 ml all the day and at evening we inoculate in 50 ml and we leave at 37°C O/N.
  • We don’t observe any colony for R0051 and I13507 and for the 2 ligations.

GFP induction test in J04431 transformed cells: we preinoculate in 5 ml. At OD: 0.4/0.6 + IPTG, we put a drop on the micro slide. Yet after 30 minutes the cells are green.

  • We harvest them and we put them in 5 ml without IPTG to see the extinction time of the protein.
  • We read every 20 minutes.
  • We get along with another GFP induction: yet after 10 minutes the cells are green.
  • We transform bacteria with 4 ul of I13507 and R0051 and with 10 ul of J04500 + J04631 (I763004) ligations.
  • We strake on plates after we have harvested.

  • We get along with Miniprep for C0051:

-Quantification: conc. 65 ng/ul;

-Cleanness n.:1.8.

  • We observe colony for I13507 and ligation. We preinoculate in 5 ml and at evening in 50 ml.
  • We don’t find any colony for R0051.
  • We transform with all the R0051 we have and alternatively with R1051.

  • We leave colonies for R0051 and for R1051 at 37°C during the day. We inoculate in 5 ml at evening and we leave them O/N.
  • We get along with fluorescence test of J04431 and J04500+J04631 (I763004) ligation to test the ligation protocoll.
  • Then we inoculate in 5 ml O/N.

  • We get along with Miniprep for R0051 and R1051. We don’t try to quantificate with spectofotometer because it isn’t sensible enough. Midi for I13507:

-Quantification: conc.:56 ng/ul.

  • Digestion for :

-I13507 with XbaI/PstI;

-R0051 with SpeI/Pst1;

-C0051 with XbaI/PstI.

  • Then we extract from gel.
  • We observe green fluorescence in J04500+J04631 (I763004) cells and in IPTG not yet inducted J04431 cells. To explain this unespected observation, we decide to identify the istant during the bacterial growth when the fluorescence appears to understand if fluorescence appearance is determinated by glucose depletion in the culture medium and/or by a deficient LacI quantity inside the cell. Infact the indogenous LacI in coli is sufficient for an only Plac and we suspect it can’t repress the high copy number plasmid Plac we have used. (We will perform this test on 31/07)