Week 4

From 2007.igem.org

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::'''07/24/07'''   
::'''07/24/07'''   
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*We observe colonies for [http://partsregistry.org/Part:BBa_C0051 C0051] plates. We inoculate in 50 ml and we leave at 37°C O/N.
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*We observe colonies for [http://partsregistry.org/Part:BBa_C0051 C0051] plate. We inoculate in 50 ml and we leave at 37°C O/N.
*We don’t observe any colony for [http://partsregistry.org/Part:BBa_R0051 R0051],[http://partsregistry.org/Part:BBa_I13507 I13507] and for the 2 ligations.
*We don’t observe any colony for [http://partsregistry.org/Part:BBa_R0051 R0051],[http://partsregistry.org/Part:BBa_I13507 I13507] and for the 2 ligations.
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'''GFP induction test''' in [http://partsregistry.org/Part:BBa_J04431 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.
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'''GFP induction test''' in [http://partsregistry.org/Part:BBa_J04431 J04431] transformed cells: we inoculate 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 and put them in 5 ml without IPTG to see the extinction time of the protein.
*We harvest and put them in 5 ml without IPTG to see the extinction time of the protein.
*We measure OD every 20 minutes.
*We measure OD every 20 minutes.

Revision as of 13:29, 3 September 2007

07/23/07
  • We observe the wrong growth of I13507 on the plate and none of R0051.
  • We get along with:

- another transformation for 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 transform 4 ul from the ligation reaction.


07/24/07
  • We observe colonies for C0051 plate. We inoculate in 50 ml and we leave at 37°C O/N.
  • We don’t observe any colony for R0051,I13507 and for the 2 ligations.

GFP induction test in J04431 transformed cells: we inoculate 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 and put them in 5 ml without IPTG to see the extinction time of the protein.
  • We measure OD 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.


07/25/07
  • 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.



07/26/07
  • 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.



07/27/07
  • 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) and J04431 cells not yet induced with IPTG. So, we decide to identify the moment in the bacterial growth when the fluorescence appears to understand if fluorescence is caused by glucose depletion in the culture medium and/or by a low LacI amount inside the cell. Infact the endogenous LacI in E. Coli is sufficient for one Plac copy and we suspect it can’t repress Plac from the high copy number plasmid we have used. (We will perform this test on 31/07)



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