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Notes from reading
- Reshma 17:46, 7 July 2006 (EDT): I forgot to mention that you should be sure and do your safety training. See Knight:Safety for details on training.
Chemically competent cells
- Reshma 19:46, 10 July 2006 (EDT): Hey, one tip ... I would name your pages something like Helene Nguewou/Grow strains rather than Grow strains. That way the page is clearly identified as yours and other won't edit it. Pages with common names like Grow strains are generally used for shared protocols on OWW. I can show you how to move pages if you like.
- Reshma 10:46, 19 July 2006 (EDT): Note that you can also do your sequence analysis by using the Registry ... see Registry sequence analysis for details.
- Grow two 'E. coli' strains with respectively an inactive 'lac' I and active 'lac' I. Store them at [math]4^o[/math]C.
- Grow a strain of 'E. coli' with the plasmid I 6030 and yellow fluorecent promoter. Store it at 4 ° C.
- Make the TSS buffer
- Grow overnight culture from the strains.
- Do a Miniprep on the I6030 cells to obtain lots of plasmid DNA.
- Prepare Chemically competent cells from the cultures of 'E. coli.
- Transform my chemically competent cells to incorporate the plasmid DNA
- Extra step added to make sure that the chemically competent cells with the piece of plasmid DNA I 6030 that we grew were not mixed up with some other cells: grow an overnight culture of the cells in a media, and perform a miniprep to obtain the DNA for a sequencing. Compare the results to the actual DNA sequencing of the expected cells.
- Make M9 agar plates with glucose, lactose and glycerol.
- Grow strains of the modified bacteria in the plates.
The following steps (instead of 9 and 10) use serial dilution and growing bacteria in various culture media in tubes.
- Use a pipet to distribute a colony of each strand in 1mL of DI water .
- Prepare M9 solution without agar and carbohydrates. Add antibacterial corresponding to the specific 'E. coli' resistance.
- Prepare a culture of bacteria in the M9 media
- Start the serial dilution with an initial volume of 150 mL of bacteria in the culture media, and then adding the appropriate concentration of bacteria in the carbohydrate-enriched media.
- Measure the fluorescence and absorbance using the Endy:Victor3 plate reader in Endy Lab
- Results from the DNA sequencing are here