Lab Session 31/07/2009

Team Newcastle 2009 iGEM 31-07-09 IMG 0231.JPG

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

Protocol

Jess tipping off the supernatant after spinning the overnight cultures in the centrifuge

We did a midi prep for the overnight cultures. We followed 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].

Procedure

To transfer the binding column, we used two eppendorf tubes for each culture. The volume of the eppendorfs 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 faster 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 eppendorf tube and mixed the solution well. Two eppendorf tubes from the same colony were combined.

Results

Goksel adding one of the agents needed for the plasmid midiprep

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.

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

Results for each tube are as below.

Results for BBa_C0056

We had high DNA concentration for the first tube.

The concentration of DNA: 124.6 ng/uL
The ratio of DNA concentration to protein concentration(260/280): 1.84
The ratio of protein concentration to RNA concentration(260/230): 1.73

Results for BBa_B1002

We had low DNA concentration for the second tube.

The concentration of DNA: 28.3 ng/uL
The ratio of DNA concentration to protein concentration(260/280): 1.66
The ratio of protein concentration to RNA concentration(260/230): 1.13

Results for BBa_R0077

The concentration of DNA: 53.8 ng/uL
The ratio of DNA concentration to protein concentration(260/280): 1.75
The ratio of protein concentration to RNA concentration(260/230): 1.39

20 – 21 June 2009 - Europe workshop (London)
23 – 24 June 2009 - UK iGEM meetup (Edinburgh)
23 October Practice Presentation (Newcastle)
23 October T-shirts are ready
27 October Practice Presentation (Sunderland)
27 October Poster is ready
30 October – 2 November 2009 - Jamboree (Boston)

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Team:Newcastle/Labwork/31 July 2009

From 2009.igem.org

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