IGEM:UNAM LCG/2009/Notebook/Hydrobium etli/2011/09/08

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Concentration verification

Abstract

I ran a gel to calculate the concentration of the 20 Anderson collection promoters and the pBBR1MCS-5 backcbone in order to have the information to do the ligation (to insert the promoters in the backbone). The promoters were ok but the Backcbone did not appear in the gel, I will need more backbone (more concentrated).

Details

I ran 2 gels because I needed more lines than the number of lines that you can put in a single gel.

Reactive ammounts

  • Concentration: Agarose 1%
  • 2μL dye + 3μL sample
  • 2μL ladder Fermentas O'Gene Ruler ready-to-use 100-10,000bp.
  • Run: 40 min, 100 volts

Results

1st gel

Lanes:

 1. Ladder
 2. J23100
 3. J23101
 4. J23102
 5. J23103
 6. J23104
 7. J23105
 8. J23106

Image:Concentracion_promoters_100-106.jpg

2nd gel

Lanes:

 1. Ladder
 2. J23107
 3. J23108
 4. J23109
 5. J23110
 6. J23111
 7. J23112
 8. J23113
 9. J23114
 10. J23115
 11. J23116
 12. J23117
 13. J23118
 14. J23119
 15. empty
 16. backbone pBBR1MCS-5

Image:Concentracion_promoters_107-119_y_backbone.jpg

Results analysis

1st gel

In this gene everything appear to go well, I just have to calculate the concentrations. The part that appear less concentrated is J23104 which I already expected. gel 03-sept

2nd gel

This second gel had more problems. First I forgot to charge the plasmid backbone, so I did it when the gel has already ran 10 minutes (so the backbone only ran 30 min). I knew that its size was aprox 4.8 Kb so I was only interested in knowing the concentration, but at the end nothing appeard in its line, I suspect that it is necessary to have it more concentrated, Helena will extract it again. And then I do not have the number of lines I was expecting, the plasmids should be from line 2 to 14 and the line 14 is empty, probably I loaded the part J23107 and the ladder in the first line, or maybe I never loaded J23119. Anyway I will have to run again a gel to see the concentration of the new plasmid backbone extraction.



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