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    Home > Biochemistry News > Biotechnology News > Preparation of X-gal solutions for commonly used storage fluids.

    Preparation of X-gal solutions for commonly used storage fluids.

    • Last Update: 2020-10-24
    • Source: Internet
    • Author: User
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    < "50%" Denhardt reagents> "50%" >Northern hybridization

    " (preparation method)

    X-gal is 5-bromine-4-chlorine-3-omega-and-beta;-D semi-lactose glycoside. Dissolve 20 mg/ml of storage liquid made with diamide dissolved X-gal. Stored in a glass tube or polypropylene tube, the test tube containing the X-gal solution shall be wrapped in aluminum foil to prevent damage due to light and shall be stored at -20 degrees C. The X-gal solution does not < need to be debacterized > aa href".

    closed agent for lowering background in hybridization tests

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    use

    hybridization using RNA probes Single-copy sequence of Southern hybridization The hybrid < of > DNA fixed to the nylon membrane Denhardt reagents are usually made with 50× storage fluids, which are filtered and stored at -20 degrees C. The storage fluid can be diluted 10 times in prebreeding fluids (often containing 0.5% SDS and 6×SSC or 6×SSPE) of denatured salmon essence DNA. The 50×Denhardt liquid contains 5g polysucrose (Ficoll, type 400, Pharmacia), 5g polyethylene pyridoxine and 5g bovineserum albumin (part V"Sigmal), plus water up to 500ml. BLOTTOGrunstein-Hogness hybridizationBenton-Davis hybridization all Southern hybridization except single-copy sequence South Hybrid Speckle Imprint 2" >1× BLOT (Bovine Lacto Transfer Technique Optimizer), a water solution containing 5% grease milk powder and 0.02% sodium laminate, should be kept at 4 degrees C. Dilute 25 times with prebreeding fluid before use. BLOTTO should not be used in the same situation as high concentrations of SDS, which can lead to themilkinfested. If the hybrid background does not meet the requirements, NP-40 to the final concentration can be added to the hybrid fluid to be 1%. BLOTTO cannot be used as a closed agent for Northern hybridization because it may contain RNA enzymes with an unacceptable level of activity. < td colspan s"2" > Note: sodium laminate is toxic, when usinggloves careful operation. Solutions containing sodium laminate should be clearly marked. < td rowspan s"2" width s"50%" > heparinSouthern hybridization in-place hybridization" heparin (Sigma H-7005, secondary product extracted from pigs or equivalent grade products) is dissolved with 4×SSPE or 4×SSC to a concentration of 50 mg/ml and is stored at 4 degrees C. Heparin is used as a closed agent in a hybrid solution containing glucosulphate at a concentration of 500 and mu;g/ml, and in a hybrid solution free of glucosulphate at a concentration of 50 and mu;g/ml. denatured and interrupted salmon DNA southern and Northern hybrid salmon sperm DNA (Sigma, III. sodium salt) dissolved in water at a concentration of 10 mg/ml and stirred at room temperature magnetism 2 to 4h if necessary. The concentration of NaCl in the solution was adjusted to 0.1mol/L and pumped once each with phenol and phenol/chloroform to recover the water phase. The combined DNA solution quickly passed through the 17th subcute injection needle 12 times to cut the DNA. Add 2 times the volume to pre-cooled ethanol to precipitate DNA. Centrifugation recovers DNA and resolubles it in water, with a concentration of 10 mg/ml, determines the OD260 value of the solution and calculates the exact DNA concentration, then boils it for 10min, which is stored in small parts at -20C. Use the front boiling water bath < a href"" > heat5min, then quickly cool down in the ice bath. The pre-hybrid solution should contain 100 samp;mu;g/ml denatured and interrupted salmon sperm DNA (Responsible Editor: King of).
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