Team:USP-UNESP-Brazil/Notebook

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{{:Team:USP-UNESP-Brazil/Templates/Header}}
{{:Team:USP-UNESP-Brazil/Templates/Header}}
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=Lab Diary=
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== <h1 id="Experiments">Experiments</h1> ==
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The participation of the people related to the Plug&Play project is show in the Experiments page, it specifies which experiment was performed by each person and the moment (weeks) that it took to be performed.
The participation of the people related to the Plug&Play project is show in the Experiments page, it specifies which experiment was performed by each person and the moment (weeks) that it took to be performed.
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https://2012.igem.org/Team:USP-UNESP-Brazil/Plasmid_Plug_n_Play/Experiments  
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[https://2012.igem.org/Team:USP-UNESP-Brazil/Plasmid_Plug_n_Play/Experiments Plug&Play Experiments]
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29/08/12 – Débora and Lucas:
29/08/12 – Débora and Lucas:
- Transformation of MR9 assembly
- Transformation of MR9 assembly
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=== 2- Hotta's and Cleslei's Lab experiments===
=== 2- Hotta's and Cleslei's Lab experiments===
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===From 12/06/2012 to 12/08/2012 – Assemblies attempts===
===From 12/06/2012 to 12/08/2012 – Assemblies attempts===
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At this period, the first attempts in order to construct the lineages that should interact in the system were made. We tried the assemblies QC1, QC6, QR4, MR1 and MR2 (see the full diagram HERE). Nothing worked properly, we had lots of fake positives or assemblies’ transformations that didn’t had any colonies. In order to fix it, we tested every single step of 3A protocols, modifying the proportions of buffer, ligase, vector and parts, assembly time, temperatures and etc. These procedures consumed a big part of our time and resources and the difficulties retarded our advancement, especially on vacations, time when we intent to accelerate the assemblies.
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At this period, the first attempts in order to construct the lineages that should interact in the system were made. We tried the assemblies QC1, QC6, QR4, MR1 and MR2 (see the full diagram [https://static.igem.org/mediawiki/2012/b/b3/Assemblys_in_english_full.jpg HERE]). Nothing worked properly, we had lots of fake positives or assemblies’ transformations that didn’t had any colonies. In order to fix it, we tested every single step of 3A protocols, modifying the proportions of buffer, ligase, vector and parts, assembly time, temperatures and etc. These procedures consumed a big part of our time and resources and the difficulties retarded our advancement, especially on vacations, time when we intent to accelerate the assemblies.
At the end of all tests, we discovered that the enzyme and buffers were, somehow, inadequate.  Were made attempts with different brands of buffer and enzymes and a new T4 ligase showed itself as good choice.
At the end of all tests, we discovered that the enzyme and buffers were, somehow, inadequate.  Were made attempts with different brands of buffer and enzymes and a new T4 ligase showed itself as good choice.
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===First assemblies confirmed===
===First assemblies confirmed===
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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.  
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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.  
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====QC1====
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====QC1====  
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12/06/2012 - Lilian, Ligation
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13/06/2012 - Daniel, Transformation
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14/06/2012 - Daniel, Inoculate
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15/06/2012 - Daniel, Miniprep
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16/06/2012 - Otto, Digestion
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17/06/2012 - Daniel, Electrophoresis Gel ([https://static.igem.org/mediawiki/2012/c/ce/QC1_e_QC6_-_2.png QC1 and QC6 Electrophoresis Gel])
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====QC6====
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12/06/2012 - Lilian, Ligation
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13/06/2012 - Daniel, Tranformation
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14/06/2012 - Daniel, Inoculum
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15/06/2012 - Otto, Miniprep
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16/06/2012 - Otto, Digestion
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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====
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06/08/2012 - Pedro, Ligation
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07/08/2012 - Pedro, Transformation
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10/08/2012 - Daniel, Inoculate
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11/06/2012 - Daniel, Miniprep
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13/06/2012 - Pedro, Digestion
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17/06/2012 - Pedro, Electrophoresis Gel([https://static.igem.org/mediawiki/2012/a/a7/QC4_120813.png QC4 Electrophoresis Gel])
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====QC5====
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14/08/2012 - Pedro, Ligation
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15/08/2012 - Daniel, Transformation
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17/08/2012 - Pedro & Daniel, Miniprep, Digestion and PCR
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18/08/2012 - Daniel, Electrophoresis Gel
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([https://static.igem.org/mediawiki/2012/b/b0/QC5_-_120818.png QC5 Electrophoresis Gel])
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====QC5==== pag:18
 
====QR4====
====QR4====
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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.
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15/05/12 – Fernando: Bacterial clones were inoculated in 5mL LB with appropriate antibiotic and grow overnight at 37C.
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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.
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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.
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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.
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04/06/12 – Fernando: Single colonies were inoculated in 5mL LB + antibiotics.
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05/06/12 – Fernando: 4,5mL from each inoculate was used for DNA extraction, the remaining 500uL was plated on LB with appropriate antibiotics.
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06/06/12 – Fernando: The 500ul plated on the day before (1D, 2M and 12M) were frozen in 20% glycerol at -80C.
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09/06/12 – Fernando: Two transformations were performed with 50ul TOP10 bacteria using 2uL miniprep from pSB1C3 and 18K (QR5).
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10/06/12 – Fernando: Single colonies from pSB1C3 and 18K were inoculated in 5mL LB + antibiotics.
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11/06/12 – Fernando: 4,5mL from each inoculate was used for DNA extraction, the remaining 500uL was plated on LB with appropriate antibiotics.
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11/06/12 - Fernando: 3A Assembly Protocol was used to assemble 12H and 12M using pSB1C3 as plasmid backbone (QR1).
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12/06/12 – Fernando: All colonies were red, showing that the assembly did not work.
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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.
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15/06/12 – Fernando: All colonies were still red, showing that the assembly did not work.
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([https://static.igem.org/mediawiki/2012/3/32/QR4.png QR4 Electrophoresis Gel])
====QR5====
====QR5====
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01/08/12 – Fernando: 3A Assembly Protocol was used to perform QR4 + 18K + pSB1C3 (QR5) assembly.
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A few changes were made to this protocol.
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- The enzyme NotI was used instead of DpnI.
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- The ligation step was performed using 4uL of digested plasmid backbone and not 2uL.
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- 2uL T4 DNA ligase was used instead of 1uL
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- No water added
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02/08/12 – Fernando: 50uL TOP10 bacteria transformed with 2uL product of the ligation step.
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06/08/12 – Fernando: Four colonies were inoculated in 5mL LB + antibiotics and grown overnight.
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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).
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([https://static.igem.org/mediawiki/2012/2/29/QR5.png QR5 Electrophoresis Gel])
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====QR6====
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08/08/12 – Fernando: 3A Assembly Protocol was used to perform QR5 + 12M + pSB1C3 (QR6) assembly.
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09/08/12 – Fernando: 50uL TOP10 bacteria transformed with 2uL product of the ligation step.
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10/08/12 – Fernando: All colonies were red.
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22/08/12 – Fernando: One more change was made in 3A Assembly Protocol. The enzyme HindIII was used instead of NotI. (This prevented the RFP coding device to ligate again to the pSB1C3 after digestion, so less red colonies were seen). 3A Assembly Protocol was used to perform QR5 + 12M + pSB1C3 (QR6) assembly.
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23/08/12 – Fernando: 50uL TOP10 bacteria transformed with 2uL product of the ligation step.
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26/08/12 – Fernando: Five colonies were inoculated in 5 mL LB + antibiotics and grew overnight.
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27/08/12 – Fernando: DNA was extracted using Quiagen Miniprep Kit.
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The QR6 construction was digested with BamHI and PstI, but the 2.363bp and 1.613bp bands could not be distinguished after gel electrophoresis.
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01/09/12 – Fernando: QR6 construction were digested again using a different set of enzymes. 3A Assembly Protocol was used to assemble 12H and 12M using pSB1C3 as plasmid backbone (QR1).
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02/09/12 – Fernando: 50uL TOP10 bacteria transformed with 2uL product of QR1 ligation step.
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03/09/12 – Fernando: All colonies were red
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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.
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([https://static.igem.org/mediawiki/2012/7/7f/QR6.jpg QR6 Electrophoresis Gel])
====QR1====
====QR1====
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18/09/2012 - Pedro - Ligation.
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19/09/2012 - Daniel & Luiza - Transformation.
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20/09/2012 - Daniel - Inoculum.
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21/09/2012 - Pedro - Digestion and PCR.
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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

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