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

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Day two. Miniprep, gel electrophoresis and gel extraction.
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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: Add H2O to 1L and adjust pH. => yield 50x desired [].
 * 242 g Tris Base
 * 57.1 mL Acetic Acid
 * 100 mL .05M EDTA

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.

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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