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In Situ Hybridization (view source)
Revision as of 15:17, 22 November 2011
, 15:17, 22 November 2011→Protocol: add solutions
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Herring Sperm DNA serves as blocker of nonspecific DNA binding +DTT (dithiothreitol) (aka Cleland's reagent) prevents oxidation of thiol groups and breaks down disulfide bonds
+
+
==Protocol==
==Protocol==
−'''DEPC-Treated Water'''
+*1 ml of DEPC per 1 L ddH2O
+*Squirt DEPC forcefully into water.
+*Cover and shake vigorously.
+*(Some protocols recommend sitting overnight.)
+*Autoclave.
−'''20x SSC'''
+''(We now purchase this from Invitrogen).
+*400 ml DEPC-H2O
+*87.65 g NaCl
+*441 g Citric Acid
+*Bring to pH 7.0
+*Bring volume to 500 ml with DEPC-H2O
+*Autoclave. (make sure volume back to 500 ml)
+
+Hybridization of DNA or RNA probes to target RNA is dependent on salt concentration (Na+ neutralizes the PO3- charges on the DNA/RNA backbones.) SSC is an old-time decoagulant that was a common buffer in early biochemical labs. The hybridization conditions are optimized for 2x SSC.
+
+
+'''Deionized Formamide:'''
+*Mix 50 ml formamide and 5 g of mixed bed ion exchange resion (Bio-Rad AG 501-X8, 20-50 mesh).
+*Stir 30 min at room temperature.
+*Filter twice thru Whatman #1 filter paper
+*Store at -20C in 15 ml tubes covered with aluminum foil.
+
+Formamide denatures nucleic acids (lowers effective Tm). Because the breakdown products of formamide degrade nucleic acids, for sensitive applications formamide should be deionized by treatment with a mixed-bed ion-exchange resin.
+
+
+'''5x TED'''
+*250 mM Tris, pH 7.4, 25 mM EDTA, 50% Dextran Sulphate
+*Weigh 3.05 g Trizma Base
+*Weigh 0.93 g EDTA
+*Add ~35-40 ml DEPC-H2O
+*Bring to pH 7.4 with HCL
+*Add 50 g Deteran Sulfate slowly, while heating slowly to boil
+*Bring volume to 100 ml with DEPC-H2O
+*Store at 4C in 15 ml tubes covered with aluminum foil
+
+Dextran is corn starch; serves as a thickener to increase effective concentration of all the other reagents.
+
+
+'''50x Denhardt's Solution'''
+''(We now purchase this from Invitrogen).
+*1% Ficoll, 1% PVP, 1% BSA
+*1 g Ficoll, 1 g PVP, 1 g BSA in 100 ml DEPC-H2O.
+*Filter.
+*Store in 1 ml tubes at -20C.
+
+Denhardt's is a mixture of high molecular weight polymers that blocks non-specific binding sites
+
+
+'''Herring/Salmon Sperm DNA'''
+''(We now purchase this from Invitrogen).
+*Dissolve 10 mg salmon or herring sperm DNA per 1 ml DEPC-H2O
+*Extraction with phenol and phenol-chloroform optional.
+*Sonicate for a few minutes until solution is no longer viscous (~3 min)
+*An OD260 to determine exact concentration of DNA is also optional.
+*Boil solution for 10 min
+*Cool on ice
+*Aliquot at 800 ul and store at -20C
+Random DNA serves as blocker of nonspecific DNA binding
+
+
+'''DTT (dithiothreitol)'''
+Aka Cleland's reagent; prevents oxidation of thiol groups and breaks down disulfide bonds
==Reagents and Solutions==
==Reagents and Solutions==