User:Paulina Alatriste/Notebook/UNAM Genomics Mexico 2011/2011/07/27

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Plasmid characterization-Stability

  • Initiation of the experiment to measure the stability of the standard plasmid.

  • Today Pablo and I started with the characterization of the standard plasmid (pBBRMCS5 with the preffix-RFP-suffik fragment). The first thing we are going to measure is the stability. So today we started with the experiment.

We previously left a culture of E. coli DH5α (which have the plasmid of interest) growing overnight in LB media with antibiotic. We want that cells grow to the stationary phase.

1. The first step consists in centrifuge all the cell content of the culture and wash the pellet with clean LB media (without antibiotic). You wash the pellet 3 times in a series of centrifugation, addition of clean media, resuspension of the pellet and centrifugation.

2.After the last wash you resuspend again the pellet in one mL of clean LB medium. This mL is transfered to another 3 mL of clean LB medium.

3. The next step is to measure the O.D of the culture, so you take one mL of the 4 you have before and put it in a cuvette for spectrophotometer. At the same time put 1 μL of clean LB medium for measure of a blank sample. After we measure our sample we obtain a O.D of 1.5819 which indicates that the cells are in a stationary phase.

4. Once we have measure the O.D we take the volume necesary for inoculate 100mL of LB media and that the new O.D of this 100mL be 0.01. The volume that we take to inoculate the 100mL was of 632μL.

5. We then shake the new culture for 1 min.

6. The next step consists in take 1 mL of this new culture (100mL) and put it in a tube. Then we diluted 100μL of the tube in 900μL of clean LB medium, of this new tube we diluted another 100μL in another 900μL of clean media, we repeat this process one more time and we obtain dilutions of 10-1, 10-2, 10-3.

7. Finally we plated petri dishes with 100μL of this 3 dilutions and left them growing for 12 hours.

  • Exactly at 9:20 p.m (twelve hours later) we have to take again another 1 mL of the remaining 99 mL. An obtain dilutions of 10-3, 10-4, 10-5 and plate again.