IGEM:PennState/Labbook/Nimrah Ahmed/2008/05/20

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Day two. Miniprep, gel electrophoresis and gel extraction.

Contents

Miniprep (a bunch of DNA in a tube)

Follow centrifuge "Spin" protocol in Miniprep Handbook in Miniprep Kit.

  1. Centrifugate bacteria: divide bacteria into aliquots, balance in centrifuge and spin with the amber-colored circular cap on for 10 minutes.
  2. Retrieve P1 from 4ºC fridge, P2 and P3 from miniprep kit. Add to each vial after dumping supernate (in autoclave bag!).
  3. Pipette into filter columns and centrifuge for 10 minutes.
  4. Dump leftover fluid (in autoclave bag!)
  5. Centrifuge with 750μL PE buffer for 30sec.
  6. Dump leftover fluid (in autoclave bag!)
  7. Centrifuge again.
  8. Save vial.

Making TAE Buffer

Need:

  • 242 g Tris Base
  • 57.1 mL Acetic Acid
  • 100 mL .05M EDTA

Add H2O to 1L and adjust pH. => yield 50x desired [].

Making Gel

  1. Mix TAE buffer with Agar 1:5.
  2. Heat in microwave 45s twice, until clear & just starting to bubble.
  3. Pour diluted (not agar) buffer into chamber under gel plate.
  4. Fit gel plate in, flush w/top.
  5. Pour heated gel Agar-Buffer solution into gel plate. Fit well comb on top: large wells for more DNA (or clumsier work).
  6. When gel hardens, reorient gel plate so that wells are on the left side.

Add other ingredients for gel electrophoresis to each vial

Ladder

  • 5x concentrated
  • Pick ladder based on expected DNA size.
  • For 1 plasmid, 1kBase ladder should be ok.
  1. Pipet in several microliters

Dye

  • 10x concentrated
  • We use Sybergreen
  • Be careful! Sybergreen is light-sensitive. Cover vials with foil at all times!
  1. Pipet in 5μL
  2. Allow dye to "soak in" for five minutes in a drawer (no light!)

Running Gel

  • Add only a little DNA (like 5μL) if you're only testing something.
  • If you're not sure the voltage is on, check sides to see if bubbles appear in buffer.
  • Run gel with foil covering.

Gel Extraction

  • Wear face shield
  1. Observe gel briefly in UV machine: check for clarity of ladder, and whether your samples migrated as expected.
  2. Open UV machine door and force the lamp on by fixing the door-switch in place with a pen.
  3. Using a sharp razor blade, quickly cut out the segment of DNA and remove to a centrifuge vial.
  4. ...I forgot there was a manual. Just follow protocol in the manual.

--Nimrah Ahmed 15:26, 22 May 2008 (EDT)

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