User:Laura Flynn/Notebook/Experimental Biological Chemistry/2011/09/21

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Objective

The purpose of this experiment was to determine if the synthesis of mutated GFP DNA was successful using gel electrophoresis.


Procedure

  • 1% Agarose Gel was prepared by mixing 25 mL of tris acetate ethylene tetracetic acid buffer with 0.25 grams of agarose. This solution was microwaved for about 40 seconds and the poured into the electrophoresis chamber and set to cool for 20 minutes. After it was properly cooled, the comb and wedges were removed and the gel was covered in the buffer that was used previously.
  • 1μl of the sample was combined with 5μl of gel loading dye on paraffin wax, and then was pipetted up and down to ensure proper distribution. A gel ladder consisting of DNA ladder, glycerol, and blue dye was loaded in well 1, and then each 6μl sample of protein and loading dye was distributed to each well. Well 3 contained the sample prepared for this experiment.
  • After all samples were loaded, the gel electrophoresis machine was turned on and set to 80 volts. It ran for about 40 minutes before it was turned off and the gel was removed from the machine. The gel was then stained with a mixture of TAE and dilute ethidium bromide for 10 minutes. It was rinsed with a mixture of TAE and then observed under UV light.


Results

DNAgel.jpg

DNA was shown present in well 3, which is where the DNA from this experiment was placed. The mark is shown around the 3000 bp mark, indicating that the correct cutting occurred in the DNA.

Analysis of the DNA sequence was conducted at an off-site sequencing business, and it was shown that there was no mutation in the DNA, as it was identical to the initial DNA.

Gel ladder: 500 bp, 1000 bp, 2000 bp, 3000 bp, 4000 bp, 5000 bp, 6000 bp, 7000 bp, 8000 bp, 9000 bp, 10000 bp.

Notes