IGEM:PennState/2006/dnagels: Difference between revisions

From OpenWetWare
Jump to navigationJump to search
No edit summary
Line 11: Line 11:
#Swirl to mix.
#Swirl to mix.
#Cover flask w/ Reynolds wrap & microwave for approx. 45 sec or until solution begins to boil.   
#Cover flask w/ Reynolds wrap & microwave for approx. 45 sec or until solution begins to boil.   
#While allowing flask to cool, assemble gel apparatus by taping ends of tray (flush against bottom to prevent leakage) and inserting combs (20-well comb holds 12+ μL). 
#Cool flask until it is hold-able.  Note: >55C may warp the tray.
#Cool flask until it is hold-able.  Note: >55C may warp the tray.
#Add 50 μL EtBr, swirl to mix, and pour in tray & allow to solidify (15 min). 
#Pour into gel box and allow to solidify, don't forget the combs!
#Remove combs, put tray in electrophoresis apparatus, fill with appropriate buffer until gel is fully submerged


===<font color="green">Loading Gel:</font>===
===<font color="green">Loading Gel:</font>===
#Add 5 μL/ladder to gel (found in fridge H)
#Add x10 SYBR green to DNA and incubate for 15 in dark
#Add x5 loading buffer
#Add 2 μL loading buffer to each sample and load on gel
#Add 2 μL loading buffer to each sample and load on gel
#Run at 100 V (if switch is on low, read lower scale; don’t go too far over or gel will melt), until bands migrate approx. 2/3 down gel.  (takes around 45 min)
#Add samples to wells being care not to spill over into adjacent wells
''
#Run at 100 V until bands migrate approx. 2/3 down gel.  (takes around 45 min)
 
[http://openwetware.org/wiki/IGEM:PennState Main]

Revision as of 11:49, 22 May 2008

AGAROSE (DNA) GEL PROTOCOL

Media/Reagents

  • 1X TAE or 1X TBE
  • Agarose (powder)
  • Ethidium Bromide (EtBr)

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)
Retrieved from "https://openwetware.org/mediawiki/index.php?title=IGEM:PennState/2006/dnagels&oldid=207269"