User:Daniel Ramirez/Notebook/UNAM Genomics Mexico 2011/2011/08/03

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χρόνος πέρασμα August 3rd 2011

B0015 Characterization

  • The J04450-E0430 transformation success cannot be determined due to technical difficulties, more time in incubation will be needed to wait for the colonies to turn red. Ligation of the bioparts J04450 and E0422.

  • Today I checked the transformation I did yesterday. Unfortunately, somehow the incubator changed its temperature to 25°C so the E. coli growth rate became slower, anyhow several colonies appeared, but all of them were gray. The biopart J04450 possesses a promoter that is regulated by the LacI operator, DH5α strain is λ- so it doesn't have the protein LacI, so the promoter behaves as if it was constitutive. The colonies turn red after 18 hours of the transformation, so adding to the slow growing, it is difficult yet to see if the ligation was succesful, I will let the petri dishes more time incubating to see if some of the colonies turn red.
  • I put to ligate another construction, biopart J04450 (the one isolated from the low agarose melting point electrophoretic gel) and E0422 (CFP). In fact, this is the most important construct as it possesses the two fluorescent proteins with the most extreme absorption and emission wavelengths, this particularity will prove very easy to measure with the spectrophotometer. The ligation was performed just as the last one:

H20 4 μl
T4 Buffer 10x 2 μl
Vector (pSB1A2 carrying E0422) 3 μl
Insert (J04450) 10 μl
T4 Ligase 1 μl
Total 20 μl