Seth Kasowitz: New page: '''Michael J. O'Neill Lab''' University of Connecticut Department of Molecular and Cell Biology Back to O'Neill Lab Protocols In So...

Seth Kasowitz — Fri, 18 May 2007 11:19:46 GMT

New page: '''Michael J. O'Neill Lab''' University of Connecticut Department of Molecular and Cell Biology <small>Back to O'Neill Lab Protocols</small> In So...

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'''[[Oneill_Lab|Michael J. O'Neill Lab]]'''
University of Connecticut Department of Molecular and Cell Biology

<small>[[Oneill_Lab:Protocols|Back to O'Neill Lab Protocols]]</small>

In Southern analysis, DNA is transferred to a membrane and then hybridized to a probe sequence. There are many variations to how this experiment is run. This protocol is a standard experiment running digested genomic DNA on a gel prior to blotting.

==Day 1==
*Setup overnight digest of 11 μg DNA.

==Day 2==
#Check for complete digestion with a small portion (<=1 μg) on a 0.8% agarose gel
#Load remaining sample on a 250ml 0.8% 1x [[Oneill_Lab:Chemicals#20x_TBE-modified|TBE]] agarose gel. Gel should be poured with a wide, thin comb.
#Electrophorese overnight at 30V. If using [[Oneill_Lab:Chemicals#10x_BlueJuice|Blue Juice]] loading dye, dye bands should split gel into equal thirds.
#*The blue dye band is at approximately 400bp
#*The green dye band is at approximately 6000bp

==Day 3==
#Save image of gel prior to blotting
#Prepare the [[Oneill_Lab:Chemicals#Blotting_Solutions|wash solutions]]
#*Depurination
#*Denaturation
#**Must be made in a plastic container.
#*Neutralization
#In a plastic tray, rinse gel briefly with water to remove excess buffer and ethidium bromide
#Remove water and rinse gel with depurination solution. Rock gently for 15 minutes
#Remove depurination solution, rinse gel with deionized water.
#Wash gel in denaturation solution, rocking for 45 minutes.
#Remove denaturation solution, and rinse gel with deionized water.
#Wash gel in neutralization solution for 15 minutes
#Remove solution and add a 2nd volume of neutralization solution. Continue to wash for 15 more minutes.
#Remove gel to a second tray and rinse with water.
#Prepare Southern apparatus
##Cut 3 pieces of Whatmann and 1 piece of Hybond-N+ equal to the size of the gel.
##Cut a wick of Whatmann long enough to drape over a glass plate such that the ends will be submerged, and 1/4 inch wider than the gel.
##Add about 1 inch of [[Oneill_Lab:Chemicals#20x_SSC|20x SSC]] to a deep plastic tray.
##Place a clean glass plate over top of tray.
##Wet wick with 20x SSC and drape over glass plate. Using a glass pipet, roll out bubbles.
##Place gel upside down on the wick, roll out bubbles.
##Mark Hybond-N+ membrane and place on top of gel. Note orientation of marking to gel. Roll out any bubbles
##Use cling wrap to place a narrow border around the membrane. Only about 3mm should be covered on each side.
##Wet a piece of Whatmann with 20x SSC and place over membrane. Roll out bubbles
##Repeat with other two pieces of Whatmanns.
##Stack paper towels on top of Whatmanns.
##Place a tray on top of paper towels.
##Use a water filled falcon tube as a weight on top of the tray
##Leave overnight

==Day 4==
#Disassemble apparatus, but leave gel and membrane attached.
#Use an erasable pen to mark well positions on membrane.
#Wet a piece of Whatmman larger than gel with 0.4M NaOH.
#Place membrane on top of Whatmman, DNA side up.
#Crosslink for 20 minutes.
#Let membrane dry on Whatmann, DNA side up.

Oneill Lab:Southern Blot - Revision history

Seth Kasowitz: New page: '''Michael J. O'Neill Lab''' University of Connecticut Department of Molecular and Cell Biology Back to O'Neill Lab Protocols In So...