Team:USP-UNESP-Brazil/Notebook

From 2012.igem.org

(Difference between revisions)
(Assembly experiments)
(From 12/06/2012 to 12/08/2012 – Assemblies attempts)
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===Assembly experiments===
===Assembly experiments===
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====From 12/06/2012 to 12/08/2012 – Assemblies attempts====
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===From 12/06/2012 to 12/08/2012 – Assemblies attempts===
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.
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 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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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.
====First assemblies confirmed====
====First assemblies confirmed====

Revision as of 20:48, 26 September 2012