Team:USP-UNESP-Brazil/Notebook

From 2012.igem.org

(Difference between revisions)
(QC5)
(First assemblies confirmed)
 
(9 intermediate revisions not shown)
Line 272: Line 272:
===First assemblies confirmed===
===First assemblies confirmed===
-
After this messy period trying to fix the assembly's problems, the first good results came. We made with fully success QC1, QC4, QC5, QR4, QR5 and QR1 assemblies through 3A protocol. The results and Agarose Gel photos follows.  
+
After this messy period trying to fix the assembly's problems, the good results came. We made with fully success QC1, QC4, QC5, QR4, QR5, QR6 and QR1 assemblies through 3A protocol. The results and Agarose Gel photos follows.  
Line 286: Line 286:
16/06/2012 - Otto, Digestion
16/06/2012 - Otto, Digestion
-
17/06/2012 - Daniel, Electrophoresis Gel
+
17/06/2012 - Daniel, Electrophoresis Gel ([https://static.igem.org/mediawiki/2012/c/ce/QC1_e_QC6_-_2.png QC1 and QC6 Electrophoresis Gel])
 +
 
 +
====QC6====
 +
12/06/2012 - Lilian, Ligation
 +
 
 +
13/06/2012 - Daniel, Tranformation
 +
 
 +
14/06/2012 - Daniel, Inoculum
 +
 
 +
15/06/2012 - Otto, Miniprep
 +
 
 +
16/06/2012 - Otto, Digestion
 +
 
 +
17/06/2012 - Daniel, Electrophoresis Gel([https://static.igem.org/mediawiki/2012/c/ce/QC1_e_QC6_-_2.png QC1 and QC6 Electrophoresis Gel])
====QC4====
====QC4====
Line 299: Line 312:
13/06/2012 - Pedro, Digestion
13/06/2012 - Pedro, Digestion
-
17/06/2012 - Pedro, Electrophoresis Gel
+
17/06/2012 - Pedro, Electrophoresis Gel([https://static.igem.org/mediawiki/2012/a/a7/QC4_120813.png QC4 Electrophoresis Gel])
====QC5====  
====QC5====  
Line 309: Line 322:
18/08/2012 - Daniel, Electrophoresis Gel
18/08/2012 - Daniel, Electrophoresis Gel
 +
([https://static.igem.org/mediawiki/2012/b/b0/QC5_-_120818.png QC5 Electrophoresis Gel])
 +
 +
-
====QC6====
 
-
12/06/2012 - Lilian, Ligation
 
====QR4====
====QR4====
 +
14/05/12 – Fernando: Bacterial transformation with 12C (QR4), 12H (QR1) and 14A (QR4) parts. Bacterial clones were plated on LB plates with appropriate antibiotic and grown overnight at 37C.
 +
 +
15/05/12 – Fernando: Bacterial clones were inoculated in 5mL LB with appropriate antibiotic and grow overnight at 37C.
 +
 +
16/05/12 – Fernando: 500ul from each inoculate was plated on LB plates with appropriate antibiotics. The remaining volume was used for DNA extraction, using Quiagen Miniprep Kit.
 +
 +
17/05/12 – Fernando: Bacterias carrying biobricks 12C, 12H and 14A, were frozen on 20% glycerol at  -80C for further use. Parts 12H and 14A were confirmed after gel electrophoresis.
 +
 +
03/06/12 – Fernando: Three transformations were made using 50ul TOP10 bacteria and 2uL miniprep volume from 1D (QR3), 2M (QR2) and 12M (QR1) parts and plated on LB with appropriate antibiotics.
 +
 +
04/06/12 – Fernando: Single colonies were inoculated in 5mL LB + antibiotics.
 +
 +
05/06/12 – Fernando: 4,5mL from each inoculate was used for DNA extraction, the remaining 500uL was plated on LB with appropriate antibiotics.
 +
 +
06/06/12 – Fernando: The 500ul plated on the day before (1D, 2M and 12M) were frozen in 20% glycerol at -80C.
 +
 +
09/06/12 – Fernando: Two transformations were performed with 50ul TOP10 bacteria using 2uL miniprep from pSB1C3 and 18K (QR5).
 +
 +
10/06/12 – Fernando: Single colonies from pSB1C3 and 18K were inoculated in 5mL LB + antibiotics.
 +
 +
11/06/12 – Fernando: 4,5mL from each inoculate was used for DNA extraction, the remaining 500uL was plated on LB with appropriate antibiotics.
 +
 +
11/06/12 - Fernando: 3A Assembly Protocol was used to assemble 12H and 12M using pSB1C3 as plasmid backbone (QR1).
 +
 +
12/06/12 – Fernando: All colonies were red, showing that the assembly did not work.
 +
 +
14/06/12 – Fernando: 3A Assembly Protocol was used to assemble 12H and 12C using pSB1C3 as plasmid backbone and also 12C and 14A, with pSB1C3 as backbone (QR4). This time newer enzymes were used.
 +
 +
15/06/12 – Fernando: All colonies were still red, showing that the assembly did not work.
 +
([https://static.igem.org/mediawiki/2012/3/32/QR4.png QR4 Electrophoresis Gel])
====QR5====
====QR5====
Line 327: Line 371:
07/08/12 – Fernando: Each inoculate was used for DNA extraction. 3uL plasmid from each of the four colonies were digested with EcoRI and PstI enzymes. QR5 assembly was confirmed with gel electrophoresis (three out of the 4 colonies were confirmed).
07/08/12 – Fernando: Each inoculate was used for DNA extraction. 3uL plasmid from each of the four colonies were digested with EcoRI and PstI enzymes. QR5 assembly was confirmed with gel electrophoresis (three out of the 4 colonies were confirmed).
 +
([https://static.igem.org/mediawiki/2012/2/29/QR5.png QR5 Electrophoresis Gel])
 +
====QR6====
====QR6====
Line 351: Line 397:
20/09/12 – Fernando: Since 3A Assembly Protocol was not working to assemble the QR1 construct, each part (12H, 12M and pSB1C3) were digested using the 3A Assembly Protocol digestion procedure and the total volume was loaded in a electrophoresis gel. The idea was to purify the inserts and linearized plasmid backbone pSB1C3 using Quiagen Gel Extraction Kit. Since the bands could barely be seen under U.V, nothing was purified.
20/09/12 – Fernando: Since 3A Assembly Protocol was not working to assemble the QR1 construct, each part (12H, 12M and pSB1C3) were digested using the 3A Assembly Protocol digestion procedure and the total volume was loaded in a electrophoresis gel. The idea was to purify the inserts and linearized plasmid backbone pSB1C3 using Quiagen Gel Extraction Kit. Since the bands could barely be seen under U.V, nothing was purified.
 +
([https://static.igem.org/mediawiki/2012/7/7f/QR6.jpg QR6 Electrophoresis Gel])
====QR1====
====QR1====
-
18/09/2012 - Daniel & Luiza - Ligation.  
+
18/09/2012 - Pedro - Ligation.
 +
 
 +
19/09/2012 - Daniel & Luiza - Transformation.
 +
 
 +
20/09/2012 - Daniel - Inoculum.
 +
 
 +
21/09/2012 - Pedro - Digestion and PCR.
 +
24/09/2012 - Pedro - Electrophoresis Gel.([https://static.igem.org/mediawiki/2012/9/91/QR1_120924b.png QR1 Electrophoresis Gel])

Latest revision as of 03:54, 27 September 2012