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Line 5: − 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 + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +
In Situ Hybridization (view source)
Revision as of 14:17, 22 November 2011
, 14:17, 22 November 2011→Protocol: add solutions
==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==