User:Paulina Alatriste/Notebook/UNAM Genomics Mexico 2011/2011/06/27

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Plasmid Isolation using a Roche Kit

ABSTRACT

  • Isolation of pBBRMCS5 plasmid from BL_21(DE3) E. coli cells using a Kit.

  • After all our attempts to extract plasmid by the conventional method, we decided to use a Kit. For this purpose we use the "High Pure Plasmid Isolation Kit" of Roche. We follow the steps indicated in the Instruction manual, pages 8 and 9. You can see this steps in the following link: Instruction Manual.

The cultures used grow 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:

Lanes:

1. Ladder 10 Kb.
2. Pablo isolation 1.
3. Pablo isolation 2.
4. My isolation 1.
5. My isolation 2.
6. Ladder 10 kb.

As you see the plasmid is very clean, but the samples are at very low concentration. One possible explanation for this is that we perform centrifugations at 13000rpm and not 13000g in the filtration steps, so maybe we lose some DNA in the process. Pablo performed a test digestion to see if ApaI enzyme is cutting the DNA. If you want to see this experiment go to the following link: Digestion test

GARDENING

  • Analysis of the plants

To see what I did about this topic please refer to my team partner Daniel notebook: Gardening



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