Haynes Lab:Notebook/Jan/2013/08/12

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Restriction Digest and Gel Electrophoresis

  • I followed the Haynes Lab protocol Assembly 101, although I did not have a second BioBrick to connect to the first one; I cut the first BioBrick part at the EcoRI and SpeI parts for practice and hoped to obtain a gel electrophoresis image of the plasmid at ~189 bp.
  • 1. The miniprep was done before today, and the plasmid used was KAH47/JDS47 at a concentration of 22 ng/μl.
  • 2. The reaction was set up as follows:

Plasmid DNA 25.0 μl* Fermentas FastDigest enzyme 1 1.0 μl Fermentas FastDigest enzyme 2 1.0 μl 10x FastDigest buffer + green loading dye 3.0 μl dH2O 0.0 μl

 	                                       30.0 μl total

The maximum amount of plasmid DNA was used and no dH2O was used because of the low concentration of the plasmid DNA (22 ng/μl).

  • 3. The tube was flicked to mix the solution thoroughly and was incubated at 37°C for 10 minutes.
  • 4. Made an agarose gel by adding 0.6 g agarose to ~60 ml 1x TAE buffer in a glass flask.
  • 5. Mixed by swirling and microwaved for 40 seconds. Mixed by swirling a second time and microwaved for 40 seconds.
  • 6. Set up a gel mold and comb.
  • 7. Added 6 μl of 10 mg/ml ethidium bromide to the agarose gel for a final concentration of ~1.0 μg/mL etBr. Mixed by swirling.
  • 8. Poured the gel into the gel mold and allowed it to cool until it became sufficiently hard.
  • 9. Filled the gel electrophoresis chamber with 1x TAE, removed the comb from the gel, and submerged the gel into the filled electrophoresis chamber.
  • 10. Pipetted 15 μL pre-made 1 kb ladder mix into the first empty well and the plasmid DNA sample into the first empty well.
  • 11. Connected the electrical leads so that the positive end is at the bottom. Ran the gel at 100 V for an hour.
  • Unfortunately, no image was obtained, as user error in pipetting the ladder into the wells resulted in a messy image. The primary purpose of this experiment was to familiarize myself with the restriction digest and gel electrophoresis process.