IGEM:UNAM-Genomics Mexico/2009/Notebook/H2 Pae/2011/08/12

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Objectives

  • To see the results of the conjugation, which will be succesful if the colinies which were re-cultivated didn't grow in LB medium
  • To do the Uriel's instructions concerning to the Gibson's test.

Results of the conjugation

Today I saw the results of the petri dishes of both PY an LB medium, all the 26 colonies wihch were re-cultivated just grew on PY medium with double antibiotic(gentamicin & nalilixic acid) indacating that all of them are R. etli colines. Our plasmid doesn't contains only the gentamicin resistance which was already aquired by the R. etli colonies, it also contains a RFP as reporter hence the colonies should be red, instead some colonies are a little bit pink. I took only one colonie (the most pink) and I re-cultivated it on PY medium with double antibiotic and CaCl2. In around three days I wil sse if the colonies are red.

Uriel's intructions

I have to see if the Gibson assembly whic was done by Uriel is succesful. To do it he said to me that he had left two petri dishes in the 30°C incubator, one containig colonies of the the assembly of a "linearized" plasmid with chloramphenicol resistance with an insert containing kanamicin resistance, the other petri dish contained colonies of a control of the assembly which was a reaction with only the "linearized" plasmid. In both of the pteri dishes was a lot of colonies indicating that the plasmid was not properly linearized; in order to see if our resistance cassette was introduced in the plasmid backbone I did the following procedure:

  • I took fifteen colonies of each petri dish and I put them in liquid medium with half of the usual concentration of kanamicin.
  • I suposed to do plasmid extraction an deigestion, but until now none of colonies have grown.