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Lab Session 31/07/2009

Previously

cinR ([http://partsregistry.org/Part:BBa_C0077 BBa_C0077]) and cinI ([http://partsregistry.org/Part:BBa_C0076 BBa_C0076]) coding sequences did not grow in LB + Kan media but they grew in plain LB media. We had overnight cultures prepared for CinR sensitive promoter([http://partsregistry.org/Part:BBa_R0077 BBa_R0077]), cI coding sequence([http://partsregistry.org/Part:BBa_B1002 BBa_B1002]) and the double terminator([http://partsregistry.org/Part:BBa_B1002 BBa_B1002]).

Today

We will do a midi prep for the overnight cultures. We will follow the protocol from GenElute ([http://www.sigmaaldrich.com/etc/medialib/docs/Sigma/Bulletin/na0200bul.Par.0001.File.tmp/na0200bul.pdf NA0200S_NA0200]). The list of plasmid kits can be accessed from [http://www.sigmaaldrich.com/life-science/molecular-biology/dna-and-rna-purification/hp-plasmid-kits.html Gen Elute's plasmid kits page].

To transfer the binding column, we used two epindorfs for each culture. The volume of the epindorfs were 450ul and we used 45ul of sodium acetate buffer and 315ul of isopropanol for "DNa concentration" step. We centrifuged the tubes at 18000rpm for 30 minutes before adding ethanol to rinse the DNAs. We then centriguged the tubes again for 20 minutes at 13000rpm. If we had used the other machine we would centrifuge the tubes at 18000rpm for 10 minutes. Since it was a slower machine, we used 20 minutes instead.

At the end we air-dried the pellets until the ethanol evaporated. We then added 50ul of PCR water to each epindorf tube and mixed the solution well. Two epindorf tubes with the same colony were combined.

Finally we measured the concentration of the DNA in the tubes using spectrometer. We used a sensitive pipet to wash the machine wth 2ul of water twice. We then loaded and measured the concentration for each tube. Between the steps we cleaned the machine with tissues. At the end we loaded with water again for a final cleaning procedure. Results for each tube are as below:

Tubes with plasmid DNAs were then placed to the -20C freezer