User:Elizabeth Ghias/Notebook/Experimental Chemistry/2013/04/23

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Objective

To conduct gel electrophoresis to test the activity of the DNase I attached to the enzyme.

Description

Electrophoresis Gel Preparation

  1. Add 50 µL HPLC water to each of the lyophelized buffer/DNA samles. Try to get as much dissolved as possible.
  1. Prepare a 1% agarose gel with 0.25g Agarose and 25mL TAE Buffer (microwaved together for 40 seconds).
  2. Set in the electrophoresis tray with the comb for 20+ minutes.

Running The Gel

  1. A DNA control for the gel was prepared by placing 1 uL of DNA in 4 uL of water with 1 uL of loading dye
  2. 5 uL of each sample was placed in a mini-pcr tube. 1 uL of loading dye was added to each tube
  3. 6 uL of previously prepared ladder + dye were placed in the first lane. The other lanes were then filled with the samples prepared.
  4. The lanes were loaded in the following order: 1) Ladder, 2) DNA alone 3) DNase I, 4) DNase I + 1 uL NHS Azide, 5) DNase I + 10 uL NHS Azide, 6) DNase I + 100 uL NHS Azide, 7) DNase I + 200 uL NHS Azide, 8) DNase I + 500 uL NHS Azide, 9) Hydrogel without DNase I, 10) DNA alone (repeated)
  5. Once the gel was loaded, it was run at 90V for 1 hour.
  6. The gel was removed from the gel apparatus and was stained in a solution of 20 uL ethidium bromide in 100 mL water for 10 minutes while rocking.
  7. The gel was then rinsed in distilled water for 5 minutes while rocking and then was visualized using UV light.

Data

Image:Numbered_gel_KLN.png

Order of Lanes: 1) Ladder, 2) DNA alone 3) DNase I, 4) DNase I + 1 uL NHS Azide, 5) DNase I + 10 uL NHS Azide, 6) DNase I + 100 uL NHS Azide, 7) DNase I + 200 uL NHS Azide, 8) DNase I + 500 uL NHS Azide, 9) Hydrogel without DNase I, 10) DNA alone (repeated)

Notes

  • None of the lanes appeared to be cleaved. Instead it appears they all traveled the same length. This suggests that the DNA was not active. May have been because the DNA/DNase reaction took place at room temperature, rather than 37°C. Additional tests are needed to determine the cause.



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