IGEM:UNAM-Genomics Mexico/2009/Notebook/H2 Pae/2011/06/27

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pBBRMCS-5 Isolation

Today Paulina Alatriste and I did the isolation of pBBRMCS-5, which in previously days was not good enough containing with chromosomal DNA. Now the isolation was done with a kit from Roche, there was only an issue: we achive one centrifugation at 13000 RPM instead 13000 Gs which was the correct velocity,and maybe causing the low concentration of plasmid isolated

The cultures used grew only for 2 and a half hours to avoid the excess of cell mass and polysaccharides that contaminate the samples. We did an electrophoresis gel to see the integrity of the plasmid:

The result is shown in the following gel:

1. Ladder 10 Kb.
2. My isolation 1.
3. My isolation 2.
4. Paulina's isolation 1.
5. Paulina's isolation 2.
6. Ladder 10 kb.

Pae 27 jun 2011.png

The gel shows that amount of DNA is low, but very clean =D.

Digestion's test

To see if apaI is working hence methylation is not occurring I left a digestion overnigth, with the following reagents' ratio:

H20 3 microL
Buffer 4 1 microL
apaI 1 microL
DNA 5 microL

Tomorrow I will see the result in an electrophoresis gel.