Purdue/30 June 2008

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===Problems with Bacterial Transformations===
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The experiments done on Friday, June 27 all turned out '''negative''' on the Amp plates, except for the previously transfected GFP labeled bacteria from Dr. Clase.  The GFP ''E. coli'' grew extremely well.  This proves that it is not the Amp agar causing the problem
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In the four tubes innoculated, all four show signs of bacterial growth, even the one that does not theoretically have any bacteria growing in it.  This means that either the tips we are using are contaminated (will use freshly autoclaven tips next time), the innoculation tubes are contaminated (will use freshly autoclaven tubes next time), or the media is contaminated (will set out 10 mL at 37C during the day today.  Check at end of day.)
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Due to these results, I feel it is prudent to conclude that our transformations failed miserably and the cause is due to overcompetition from wild type species accidentally included in the culturing medium for the overnight recovery.  As for the one-hour recovery, I feel that there was simply not enough time to recover from the heat shock and possibly that there was not enough DNA to fully transform the cells.
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'''Edited by Craig Barcus'''
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In lab today, we inoculated 4 tubes with a more sterile procedure.  We used a single, sterile pipet to get the media from the flask, and quickly added about 5mL to each sterile inoculation tube.  New cell cultures were taken by dipping a micropipette (autoclaved tips) into culture and then adding 25uL into the new media.
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The 4 samples included:
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#Control: LB media alone (to test media for contamination)
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#GFP strain
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#SOS "transformed" strain
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#LacZ "transformed" strain
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'''Edited by Janie Stine'''

Latest revision as of 20:23, 30 June 2008

Click Here to return to the notebook.

Problems with Bacterial Transformations

The experiments done on Friday, June 27 all turned out negative on the Amp plates, except for the previously transfected GFP labeled bacteria from Dr. Clase. The GFP E. coli grew extremely well. This proves that it is not the Amp agar causing the problem

In the four tubes innoculated, all four show signs of bacterial growth, even the one that does not theoretically have any bacteria growing in it. This means that either the tips we are using are contaminated (will use freshly autoclaven tips next time), the innoculation tubes are contaminated (will use freshly autoclaven tubes next time), or the media is contaminated (will set out 10 mL at 37C during the day today. Check at end of day.)

Due to these results, I feel it is prudent to conclude that our transformations failed miserably and the cause is due to overcompetition from wild type species accidentally included in the culturing medium for the overnight recovery. As for the one-hour recovery, I feel that there was simply not enough time to recover from the heat shock and possibly that there was not enough DNA to fully transform the cells.

Edited by Craig Barcus


In lab today, we inoculated 4 tubes with a more sterile procedure. We used a single, sterile pipet to get the media from the flask, and quickly added about 5mL to each sterile inoculation tube. New cell cultures were taken by dipping a micropipette (autoclaved tips) into culture and then adding 25uL into the new media.

The 4 samples included:

  1. Control: LB media alone (to test media for contamination)
  2. GFP strain
  3. SOS "transformed" strain
  4. LacZ "transformed" strain

Edited by Janie Stine

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