IGEM:PennState/2006/dnagels

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Current revision (14:50, 22 May 2008) (view source)
 
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*1X TAE or 1X TBE
*1X TAE or 1X TBE
*Agarose (powder)
*Agarose (powder)
-
*Ethidium Bromide (EtBr)
+
 
===<font color="green">Gel</font>===
===<font color="green">Gel</font>===

Current revision

Contents

AGAROSE (DNA) GEL PROTOCOL

Media/Reagents

  • 1X TAE or 1X TBE
  • Agarose (powder)


Gel

  1. Make [x] % agarose gel (%= weight/volume) according to size DNA you want to separate (higher MW bands are better resolved by lower percentage gels, e.g. 0.6%, while lower MW bands are best resolved by higher percentage gels, e.g. 1.4%). For most applications, 1% gel is appropriate, so weigh 1g agarose, and place in a 250 mL erlenmeyer flask (or appropriately large glassware so that the solution won’t boil over).
  2. To it add 100 mL 1X TAE or 1X TBE (TAE is used when DNA will be extracted from gel, TBE is used for diagnostic purposes).
  3. Swirl to mix.
  4. Cover flask w/ Reynolds wrap & microwave for approx. 45 sec or until solution begins to boil.
  5. Cool flask until it is hold-able. Note: >55C may warp the tray.
  6. Pour into gel box and allow to solidify, don't forget the combs!

Loading Gel:

  1. Add x10 SYBR green to DNA and incubate for 15 in dark
  2. Add x5 loading buffer
  3. Add 2 μL loading buffer to each sample and load on gel
  4. Add samples to wells being care not to spill over into adjacent wells
  5. Run at 100 V until bands migrate approx. 2/3 down gel. (takes around 45 min)
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