User:Daniel Ramirez/Notebook/UNAM Genomics Mexico 2011/2011/08/01

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χρόνος πέρασμα August 1st 2011

B0015 Characterization

  • Ligation of bioparts J04450 and E0430 to obtain the first finished construction to characterize the transcriptional terminator B0015. Also, double digestion of all the bioparts that are planned to be used (E0422, E0430, I20260 and J04450) to have a bigger stock of the bioparts.

  • Today I proceeded to assemble the first construction to characterize the transcriptional terminator B0015. This construction will consist of the plasmid backbone pSB1A2 (a high copy number plasmid carrying ampicillin resistance), the biopart J04450 followed immediately by E0430. These bioparts have been already isolated, so today I just put them to ligate with the following reactants:

H20 4 μl
T4 Buffer 10x 2 μl
Vector (pSB1A2 carrying E0430) 3 μl
Insert (J04450) 10 μl
T4 Ligase 1 μl
Total 20 μl

  • The ligation was left overnight at 25°C.
  • I also put to digest all the plasmid extractions I did friday with their proper restriction enzymes (E0422 and E0430 with EcoRI-HF and XbaI, I20260 and J04450 with EcoRI-HF and SpeI). These double digestions will serve as a stock to have a big amount of plasmid if the ligations prove to be difficult to get. The digestions were performed as follows:

H20 12 μl
Buffer #4 10x 3 μl
BSA 2 μl
EcoRI-HF 1.5 μl
XbaI or SpeI 1.5 μl
DNA 10 μl
Total 30 μl

  • The digestions were left incubating at 37°C overnight.