Uppsala-Sweden/30 July 2009

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Revision as of 19:38, 1 August 2009 by Anders (Talk | contribs)




kivd adhII pdc PCR

Today we begun the trials to extract the kivd gene from Lactococcus lactis, and adhII and pdc from S.cerivase.

We will try to extract the gene from three different strains of filmjölk, a swedish dairy product similar to yoghurt; Filmjölk and Långfil from Arla, and Verum Hälsofil from Norrmejerier. The the Filmjölk contains basic fil culture, that is Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. cremoris, Lactococcus lactis biovar. diacetylactis, and Leuconostoc mesenteroides subsp. cremoris. The Långfil is the same as Filmjölk but also includes three different strains of Lactococcus lacti longi. The Verum Hälsofil contains an unknown fil culture from Norrmejerier and also the Lactococcus lacti lacti L1A.

In preperation for the PCR 5 ml of filmjölk is washed five times with deionized water with 4000 g centrifugation in between to collect bacteria.

edit:The washing of the filmjölk failed, simple to much fats and proteins in there. Instead we performed a DNA extraction according to the protocol Protocol Ethanol-Filmjölkl.doc at [http://uppsalaigem.pbworks.com/browse/#view=ViewFolder&param=Protocols] (unavailable for outside users)

With this we managed to collect DNA from the filmjölk sample, although in with a ten times protein contamination.

We stored this on ice awaiting PCR.

--Anders 19:38, 1 August 2009 (UTC)

--Anders 13:49, 30 July 2009 (UTC)

This morning I did the finishing step in the creation of seed stock for competent E.coli as given by: [http://openwetware.org/wiki/TOP10_chemically_competent_cells ]. Depatures from protocol in this step was the use of E-tubes instead of cryo tubes for the storage of the seed stock. We'll just have to avoid dropping the frozen tubes ;)

--Anders 13:49, 30 July 2009 (UTC)

Later we inoculated 300 ml of LB medium with 1ml seed stock (e.coli at 25°C), to grow over night. This was also a lapse in the procedure, since the protocol advised the use of SOB medium. [http://openwetware.org/wiki/TOP10_chemically_competent_cells ]

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