User:Michaela Harper/Notebook/Biological Chemistry Lab/2011/09/21

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Objective

To determine whether or not the synthesis of mutated GFP DNA was successful through gel electrophoresis.

Description

  1. Add 0.25 g of 0.01 g/mL agarose to 25 mL of 1x tris base, acetic acid, and ethylenediaminetetraacetic acid (TAE) buffer. Heat the solution in a microwave for 40 seconds. The result is a 1% agarose gel used in electrophoresis. Pour the solution into the gel container with the appropriate dams. Allow the gel to cool for approximately 20 minutes.
  2. Mix 1 μL of 6x gel loading dye with 5 μL of the mutated DNA sample.
  3. Load the samples with dye mixture and a ladder containing DNA, glycerol, and gel loading dye into the wells of the gel. The ladder used was as follows: 500 bp, 1000 bp, 2000 bp, 3000 bp, 4000 bp, 5000 bp, 6000 bp, 7000 bp, 8000 bp, 9000 bp, 10000 bp.
  4. Run the gel electrophoresis at 80 V for 40 min.
  5. Remove gel and place in TAE and ethidium bromide (EtBr) solution, mix for 15 min.
  6. Rinse gel with TAE for 5 min.

Data

All groups were successful in the synthesis of the mutated DNA. The desired product was 3600 bp. Using a UV light, we were able to see the DNA of all groups located between the 3000 bp and 4000 bp ladder spots, indicating a successful synthesis. The photo of the electrophoresis gel:

DNA gel 092111 003.jpg

The first column is the ladder. The next columns are groups 1, 2, 3, 4, and 5.

Notes