User:Daniel Ramirez/Notebook/UNAM Genomics Mexico 2011/2011/07/26

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χρόνος πέρασμα July 26th 2011

ABSTRACT

  • Plasmid extraction and digestion with EcoRI-HF of the bioparts E0430 and J04450, this is to assemble the construction needed to characterize the transcriptional terminator B0015.

  • Today I extracted the plasmids that carry the bioparts E0430 and J04450 using the alkaline lysis minipreps. I followed the protocol that Miguel Ángel gave us. Lanes #2 and #3 contain the E0430 plasmid extraction, lanes #4 and #5 contain the J04450 plasmid extraction.


Dramirez plasmidextraction e0430 j04450.jpg


  • In order to see if they are indeed the plasmids that contain the bioparts, I intented to linearize them and to see their sizes. Fortunately, EcoRI restriction enzyme site found in the prefix of the bioparts is used in both parts that are to be assembled through Standard Assembly [1], so this digestion will not only help to see the plasmid sizes but also will serve to prepare the parts to be assembled. As it can be seen from the electrophoretic gel, the first E0430 extraction isn't as concentrated as the other three extractions. The digestions were performed as follows:


Reactants Very concentrated DNA Not as concentrated
H20 11 μl 9 μl
EcoRI Buffer 10x 2 μl 2 μl
EcoRI-HF 1 μl 1 μl
DNA 6 μl 8 μl
Total 20 μl 20 μl


  • The digestions were left overnight at 37°C.
  • I also helped Melissa Molho and Vladimir Muñoz in inoculating E.coli cells in 3 ml of LB with ampicillin. This bacteria carry plasmids that contain the bioparts with the different promoters from the Anderson collection.