User:Paulina Alatriste/Notebook/UNAM Genomics Mexico 2011/2011/09/04

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Assembly of parts

ABSTRACT

  • Plasmid extraction of HydA, PFOR1, and pBBRMCS5 plasmids.

  • Today Daniel helped me to extract plasmid pBBRMCS5, and the two plamids where HydA and PFOR1 sequences are inserted. For this purpose we used 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

After obtaining the samples, we did an electrophoresis with a 1% agarose gel, 130 V x 30 min. The proportions of the samples were 2μL of dye and 5 μL of plasmid DNA.

Lanes:

 1. Ladder
 2. pBBRMCS5 extraction 1
 3. pBBRMCS5 extraction 2
 4. pBBRMCS5 extraction 3
 5. pBBRMCS5 extraction 4
 6. pBBRMCS5 extraction 5
 7. PFOR 1 extraction 1
 8. PFOR 1 extraction 2
 9. PFOR 1 extraction 3
 10. PFOR 1 extraction 4
 11. PFOR 1 extraction 5
 12. HydA extraction 1
 13. HydA extraction 2
 14. HydA extraction 3
 15. HydA extraction 4
 16. HydA extraction 5

GEL EXTRACCIONES.jpg

As you see mane lanes are empty or less visible, we thought that the plasmid is very diluted so with help of a speed vac I´m going to concentrate the samples and run another electrophoresis gel.

The concentration of the samples consisted in centrifuge the tubes with the lid open in a speed vac for around 30 min. Every 10 min I checked the tubes to see the volume and when the volume was reduces to a half I stopped the process. I ran another electrophoresis gel 30 min at 130 volts:

Lanes:

 1. Ladder
 2. Ladder
 3. pBBRMCS5 extraction 1
 4. pBBRMCS5 extraction 2
 5. pBBRMCS5 extraction 3
 6. pBBRMCS5 extraction 4
 7. pBBRMCS5 extraction 5
 8. HydA extraction 1
 9. Ladder
 10. HydA extraction 2
 11. HydA extraction 3
 12. HydA extraction 4
 13. HydA extraction 5
 14. PFOR 1 extraction 1
 15. PFOR 1 extraction 2
 16. PFOR 1 extraction 3
 

HydAPFOR1ay-que-padre.jpg

As you see this time the products are visible and they are very concentrated.

  • Helena obtain another picture of the gel showing the relative concentrations of product, she needed this in order to calculate the proportions of a digestion reaction.

HistogramaHydAPFOR1Relieve.jpg