User:Paulina Alatriste/Notebook/UNAM Genomics Mexico 2011/2011/08/01

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Plasmid Characterization-Stability, continuation

ABSTRACT

  • Count of colonies of hour 60, picked of colonies of hour 72.
  • Conjugation of E. coli S17 and R. etli.

  • Today the first thing I did was to count the colonies picked and striated for hour 60. The 100 colonies in each petri dish grow well. The next thing I did was to pick colonies with a sterile stick (from the petri dishes of hour 74) and striate one little line in two petri dishes one with LB medium+gentamicin an the other with only LB media (all with the same stick). I left the petri dishes incubating at 37°C.

Conjugation

  • Because our system has to be insert in R. etli is of importance that the plasmid is also characterized in this bacteria. So the first step is to conjugate E. coli carrying our plasmid with the strain of R. etli of interest. Today I made one conjugation, the procedure is the following:

1. Put to grow in 3 μL of clean liquid PY media (without antibiotic) the strain of E. coli of interest.

2. Two or three days before put to grow R. etli in liquid clean PY media.

3. Put 200μL of the culture of R. etli in an Eppendorf tube.

4. Add to this tube 100μL of the E. coli culture.

5. Take all the content of the tube an put it in the center of a petri dish with solid and clean PY media.

6. Let the drop dry.

7. Incubate the petri dish at 30°C.


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