IGEM:UNAM-Genomics Mexico/2009/Notebook/H2 Pae/2011/07/28

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Abtract

1. I continued with the experiment of the plasmid's stability.

2. I started again the conjugation of our standard plasmid (pBBR1MCS-5) which has a RFP. This time the conjugation will be done with the canonic procedure.

3. I leaved E. coli cultive to grow in liquid medium to further extract the pBBR1MCS-5 plasmid.

Methods

Plasmid stability

To see the results of the 0 hours and 12 hours of plasmid stability refer to Paulina's Logbook on july 28

At 9:20 AM I took 1 ml of the culture which was, at this point, 24 hours old. I repeat the procedure the yesterday's procedure of:

1. Vortex the 1 ml culture.
2. Take 100μl of the previous culture and put them in 900 μL of liquid medium of antibiotic. This is the first dilution.
3. Vortex 30 times the dilution.
4. Repeat the steps 2 and 3 until reach the desired dilution.

I did it until reached the 6 dilution, becuase the original culture was 24 hour old and there were a lot of bacteria. Then I plaqued 100 μL of the dilutions 4,5 and 6.

Conjugation

  • At 4:00 PM I put to grow R. eli CE3 into 3 ml of liquid PY.
  • At 9:30 PM I put to grow E. coli S17 into 3 ml of liquid PY, this E. coli is transformated with our standard plasmid.


Extraction

At 9:35 PM I put 15 cultures of E. coli DH5α carrying our standard plasmid