Week 4

From 2007.igem.org

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::'''07/23/07'''
::'''07/23/07'''
-
*We observed the uncorrected grew of I13507 on the plate and none of R0051.
 
-
*We got along with:
 
-
- another transformation of R0051 (2 ul), I13507 and C0051;
 
-
- the ligation of J04500+J04631 (I763004) to test the ligation protocoll 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.  
+
*On the plates we observe the wrong growth of [http://partsregistry.org/Part:BBa_I13507 I13507] and [http://partsregistry.org/Part:BBa_R0051 R0051].
-
*Then we transformed 4 ul of ligate.   
+
* Transformation again for [http://partsregistry.org/Part:BBa_R0051 R0051] (2ul), [http://partsregistry.org/Part:BBa_I13507 I13507] and [http://partsregistry.org/Part:BBa_C0051 C0051];
 +
 
 +
*[http://partsregistry.org/Part:BBa_J04500 J04500]+[http://partsregistry.org/Part:BBa_J04631 J04631] ([http://partsregistry.org/Part:BBa_I763004 I763004]) ligation to test ligation protocol. We try with vector and insert 2ul, vector and insert 2-4ul. We leave it at 25°C for 1 h and at 65°C for 10 minutes.  
 +
*Then we transform 4ul from the ligation reaction.   
::'''07/24/07'''   
::'''07/24/07'''   
-
*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 observe colonies for [http://partsregistry.org/Part:BBa_C0051 C0051] plate. We inoculate in 50ml and we leave at 37°C O/N.
-
*We don’t observe any colony for R0051 and 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.
-
'''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.
+
'''GFP induction test''' in [http://partsregistry.org/Part:BBa_J04431 J04431] transformed cells: we inoculate in 5ml. At OD: 0.4/0.6 + IPTG, we put a drop on the micro slide. After 30 minutes the cells are already green.
-
*We harvest them  and we put them in 5 ml without IPTG to see the extinction time of the protein.
+
*We harvest and put them in 5ml without IPTG to see the protein extinction time.
-
*We read every 20 minutes.
+
*We measure OD every 20 minutes.
-
*We get along with another GFP induction: yet after 10 minutes the cells are green.
+
*We go along with another GFP induction: after 10 minutes the cells are already green.
-
*We transform bacteria wiht 4 ul of I13507 and R0051 and with 10 ul of  J04500 +  J04631 (I763004) ligations.
+
*We transform bacteria with 4ul of the [http://partsregistry.org/Part:BBa_I13507 I13507] and [http://partsregistry.org/Part:BBa_R0051 R0051] and with 10ul from [http://partsregistry.org/Part:BBa_J04500 J04500] +  [http://partsregistry.org/Part:BBa_J04631 J04631] ([http://partsregistry.org/Part:BBa_I763004 I763004]) ligations.
-
*We strake on plates after we have harvested.
+
*We strake on plates.
::'''07/25/07'''
::'''07/25/07'''
-
*We get along with Miniprep for C0051:
+
*Midiprep for [http://partsregistry.org/Part:BBa_C0051 C0051]:
-
-Quantification: conc. 65 ng/ul;
+
*Midiprep Quantification: conc. 65ng/ul;
-
-Cleanness n.:1.8.
+
*We observe colonies for [http://partsregistry.org/Part:BBa_I13507 I13507] and ligation. We inoculate in 50ml.
-
*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 [http://partsregistry.org/Part:BBa_R0051 R0051].  
-
*We don’t find any coloby for R0051.  
+
*We transform again with [http://partsregistry.org/Part:BBa_R0051 R0051] and with [http://partsregistry.org/Part:BBa_R1051 R1051].
-
*We transform with all the R0051 we have and alternatively with R1051.
+
Line 35: Line 34:
::'''07/26/07'''
::'''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 let colonies from [http://partsregistry.org/Part:BBa_R0051 R0051] and from[http://partsregistry.org/Part:BBa_R1051 R1051] grow at 37°C during the day. We inoculate in 5ml in the 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.
+
*We perform a fluorescence test of [http://partsregistry.org/Part:BBa_J04431 J04431] and [http://partsregistry.org/Part:BBa_J04500 J04500]+[http://partsregistry.org/Part:BBa_J04631 J04631] ([http://partsregistry.org/Part:BBa_I763004 I763004]) ligation to test the [[Bologna University/Ligation protocol |ligation protocol]].
-
*Then We inoculate in 5 ml O/N.
+
*Then we inoculate in 5ml O/N.
Line 43: Line 42:
      
      
::'''07/27/07'''
::'''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:
+
*Miniprep for [http://partsregistry.org/Part:BBa_R0051 R0051] and [http://partsregistry.org/Part:BBa_R1051 R1051]. We don’t try to quantificate with spectrophotometer because it isn’t sensible enough. Midi for [http://partsregistry.org/Part:BBa_I13507 I13507]:
-Quantification: conc.:56 ng/ul.
-Quantification: conc.:56 ng/ul.
-
*Digestion for :  
+
*Digestion for:  
-
-I13507 with XbaI/PstI;
+
-[http://partsregistry.org/Part:BBa_I13507 I13507] with XbaI/PstI;
-
-R0051 with SpeI/Pst1;
+
-[http://partsregistry.org/Part:BBa_R0051 R0051] with SpeI/Pst1;
-
-C0051 with XbaI/PstI.
+
-[http://partsregistry.org/Part:BBa_C0051 C0051] with XbaI/PstI.
*Then we extract from gel.
*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 durign the bacterial grew 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 indogene LacI in coli is sufficient for an only Plac ane we suspect it can’t repress the high copy number plasmid Plac we have used. (We will perform this test on 31/07)
+
*We observe green fluorescence in [http://partsregistry.org/Part:BBa_J04500 J04500]+[http://partsregistry.org/Part:BBa_J04631 J04631] ([http://partsregistry.org/Part:BBa_I763004 I763004]) and [http://partsregistry.org/Part:BBa_J04431 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 it is caused by glucose depletion in the culture medium and/or by a low LacI amount inside the cell. In fact 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)
 +
 
 +
 
-
[[Bologna | Back]]
+
[[Bologna#Diary | Back]]

Latest revision as of 15:50, 26 October 2007

07/23/07
  • On the plates we observe the wrong growth of [http://partsregistry.org/Part:BBa_I13507 I13507] and [http://partsregistry.org/Part:BBa_R0051 R0051].
  • Transformation again for [http://partsregistry.org/Part:BBa_R0051 R0051] (2ul), [http://partsregistry.org/Part:BBa_I13507 I13507] and [http://partsregistry.org/Part:BBa_C0051 C0051];
  • [http://partsregistry.org/Part:BBa_J04500 J04500]+[http://partsregistry.org/Part:BBa_J04631 J04631] ([http://partsregistry.org/Part:BBa_I763004 I763004]) ligation to test ligation protocol. We try with vector and insert 2ul, vector and insert 2-4ul. We leave it at 25°C for 1 h and at 65°C for 10 minutes.
  • Then we transform 4ul from the ligation reaction.


07/24/07
  • We observe colonies for [http://partsregistry.org/Part:BBa_C0051 C0051] plate. We inoculate in 50ml 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.

GFP induction test in [http://partsregistry.org/Part:BBa_J04431 J04431] transformed cells: we inoculate in 5ml. At OD: 0.4/0.6 + IPTG, we put a drop on the micro slide. After 30 minutes the cells are already green.

  • We harvest and put them in 5ml without IPTG to see the protein extinction time.
  • We measure OD every 20 minutes.
  • We go along with another GFP induction: after 10 minutes the cells are already green.
  • We transform bacteria with 4ul of the [http://partsregistry.org/Part:BBa_I13507 I13507] and [http://partsregistry.org/Part:BBa_R0051 R0051] and with 10ul from [http://partsregistry.org/Part:BBa_J04500 J04500] + [http://partsregistry.org/Part:BBa_J04631 J04631] ([http://partsregistry.org/Part:BBa_I763004 I763004]) ligations.
  • We strake on plates.


07/25/07
  • Midiprep for [http://partsregistry.org/Part:BBa_C0051 C0051]:
  • Midiprep Quantification: conc. 65ng/ul;
  • We observe colonies for [http://partsregistry.org/Part:BBa_I13507 I13507] and ligation. We inoculate in 50ml.
  • We don’t find any colony for [http://partsregistry.org/Part:BBa_R0051 R0051].
  • We transform again with [http://partsregistry.org/Part:BBa_R0051 R0051] and with [http://partsregistry.org/Part:BBa_R1051 R1051].



07/26/07
  • We let colonies from [http://partsregistry.org/Part:BBa_R0051 R0051] and from[http://partsregistry.org/Part:BBa_R1051 R1051] grow at 37°C during the day. We inoculate in 5ml in the evening and we leave them O/N.
  • We perform a fluorescence test of [http://partsregistry.org/Part:BBa_J04431 J04431] and [http://partsregistry.org/Part:BBa_J04500 J04500]+[http://partsregistry.org/Part:BBa_J04631 J04631] ([http://partsregistry.org/Part:BBa_I763004 I763004]) ligation to test the ligation protocol.
  • Then we inoculate in 5ml O/N.



07/27/07
  • Miniprep for [http://partsregistry.org/Part:BBa_R0051 R0051] and [http://partsregistry.org/Part:BBa_R1051 R1051]. We don’t try to quantificate with spectrophotometer because it isn’t sensible enough. Midi for [http://partsregistry.org/Part:BBa_I13507 I13507]:

-Quantification: conc.:56 ng/ul.

  • Digestion for:

-[http://partsregistry.org/Part:BBa_I13507 I13507] with XbaI/PstI;

-[http://partsregistry.org/Part:BBa_R0051 R0051] with SpeI/Pst1;

-[http://partsregistry.org/Part:BBa_C0051 C0051] with XbaI/PstI.

  • Then we extract from gel.
  • We observe green fluorescence in [http://partsregistry.org/Part:BBa_J04500 J04500]+[http://partsregistry.org/Part:BBa_J04631 J04631] ([http://partsregistry.org/Part:BBa_I763004 I763004]) and [http://partsregistry.org/Part:BBa_J04431 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 it is caused by glucose depletion in the culture medium and/or by a low LacI amount inside the cell. In fact 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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