IGEM:UNAM-Genomics Mexico/2009/Notebook/H2 Pae/2011/06/16

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Plasmid extraction

Paulina, Daniel and I extracted pBBRMCS-5 and pBS1T3 from E. coli DH5alhpa using this protocol, in order to digest the first one with Apa1/Sac1 and to amplify a pSB1T3 region neccesary for the standardization

We did 5 extractions(3:pBBRMCS-5, 2:PSB1T3)

The first extracion failed:

Digestion

We noticed that Apa1 doesn't work with dcm+ strains, which methylate cytosines in a specific sequence, and DH5-alfa is dcm+ so we need a strai dcm-. However we did a test doing the digestion with the plasmid from DH5-alfa. These are the proportions of the digestion:

H20 6microL
Buffer 4 1 microL
DNA 2 microL
ApaI 1microL

Overnight

Tomorrow I will do an elctrophoresis of this.



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