IGEM:UNAM-Genomics Mexico/2009/Notebook/H2 Pae/2011/07/14

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Conjugation

Once we tested that our plasmid was correctly assembled I proceeded to conjugate it to R. etli through E. coli. This was the procedure:

1. I put to grow in 1 mL of liquid PY a big pellet of: E. coli DH5α carrying the pBBR1MCS-5(donor), E. coli BL21 also carrying it(donor) and E. coli HB101(helper); the cultives were left 4 hours. It's also neccesary to have R. elti CE3 grown in this moment because the R. etli duplication is more slowly, Miguel gave me on cultive of grown R. etli.

2. In a steril test tube I put 50μL of the grown E. coli DH5α, 50μL of the grown E. coli HB101 and 100μL R. etli CE3, in another seteril test tube I did te same but with E. coli BL21 instead E. coli DH5μL.

3. I took all the 200 μL of the two test tubes and I put each of them in the center of two petri dishes with solid PY, they were left over nigth at 30°C

Tomorrow I will continue with the procedure