northern blot（Northern印迹）.doc

northern blot（Northern印迹） Northern Blot Principle and experimental method Release date: [2008-5-17] read [22450] times Principle: Under denaturing conditions, the RNA samples to be examined were subjected to agarose gel electrophoresis, followed by a rotating membrane and hybridized with a probe in accordance with the same principle of Southern and Blot. Uses: to detect whether the product contains a gene transcript (mRNA) and its content. Click to see nucleic acid hybridization products Experimental method: [instruments, reagents, materials] (I) instrument Constant temperature water bath box, electrophoresis, gel imaging system, vacuum transfer apparatus, vacuum pump, UV crosslinkers, hybrid furnace, bed temperature, decolorization table, vortex oscillator, spectrophotometer, pipette, furnace (or microwave), centrifuge tube, cylinder, beaker, flask, etc.. (two) materials Total RNA sample or mRNA sample, probe template DNA (25, NG), nylon film (three) reagent NorthernMax Kit (Cat. # 1940, Ambion, Inc., DEPC, X) agarose, optical film, film cassette, Random Primer, dNTP Mixture, 111 TBq/mmol[a-32P]dCTP, Exo-free Klenow Fragment and 10 X Buffer, Sephadex G-50, SDS, hydrogen peroxide, water sterilization etc.. [methods and procedures] 1. appliances ready: Bake at 180 degrees for atensils: cone bottles, cylinder, tweezers, blade 4 hours. Electrophoresis tank: cleaning comb and electrophoresis tank, and soak overnight with hydrogen peroxide, rinse with DEPC water, dry standby. Treat DEPC water (2 L) reserve. 2. use RNAZaP to remove the RNase enzyme pollution on the surface of tools Scrub the comb, electrophoresis slot, blade, etc with RNAZap, then rinse with DEPC water two times, remove RNAZap. 3. glue: 1., the 0.36mg agarose is added to the conical cone and 32.4mlDEPC water is added. The microwave is heated until the agarose is completely fused. 60 air bath equilibrium solution (add DEPC water to supplement evaporation water) 2. add 3.6ml 10 * Denaturing Gel buffer in th