一、实验原理 The E.Z.N.A. HP Total RNA Kit uses the reversible binding properties of HiBind matrix, a new silica-based material. This is combined with the speed of mini-column spin technology. A specifically formulated high salt buffer system allows more than 100 ug of RNA molecules greater than 200 bases to bind to the matrix. Cells or tissues are first lysed under denaturing conditions that practically inactivate RNases. Samples are then applied to the HiBind spin columns to which total RNA binds, while cellular debris and other contaminants are effectively washed away. High quality RNA is finally eluted in DEPC-treated sterile water.   二、实验试剂 Additional materials to be supplied by user 1. RNase-free Lysozyme 2. TE buffer (10mM Tris-HCl, PH7.6, 1mM EDTA )   三、实验步骤 1.  Harvest cells and resuspend in 100ul TE Buffer/lysozyme and incubate at RT for 10 minutes. Centrifuge 109 cells at 4 000 × g for 5 minute. Discard supernatant and add 100 ul of TE Buffer containing lysozyme(0.5 mg/ml for Gram-negative and 4mg/ml for Gram-positive bacteria). Resuspend cells completely and incubate at room temperature for 7 min.   2.  Add 500ul Buffer GTC/2-ME and mix by pipetting up and down several times. Remember to add 20 ul of 2-mercaptoethanol per 1 ml of Buffer GTC.   3.  Centrifuge at 14,000 x g for 5 min. Transfer the clear lysate into a gDNA Removal Column place in 2 ml collection tube. Centrifuge at 14,000 x g for 1 minute at room temperature. Transfer the flow-through lysate into a new 1.5ml tube.   4.  Add 0.5 volume of absolute ethanol (96-100%, room temperature ) to the mixture and mix well by pipetting.   5.  Add 0.5 volume (250ul or 350ul) of absolute ethanol (room temperature) to the lysate and mix well by pipetting.   6.  Apply the sample onto HiBind? RNA Mini column. The maximum capacity of the spin cartridge is 750ul. (Larger volumes can be loaded successively.) A precipitate may form on addition of ethanol in step 4. Vortex and add the entire mixture to the column. With the spin column inside a 2 ml collection tube (supplied with kit), centrifuge at 10,000 x g for 30-60 seconds min at room temperature. Discard flow-through and proceed to step 6.   7.  Place column in a new 2 ml collection tube and add 300ul RNA Wash Buffer I. Centrifuge as above and discard flow-through. Reuse the collection tube in step 7. If on-membrane DNase I digestion is desired, proceed to step 7, otherwise go to step 8.   8.  DNase I digestion (Optional) Since HiBind  RNA resin and spin-column technology actually removes most of DNA without the DNase treatment, it is not necessary to do DNase digestion for most downstream applications. However, certain sensitive RNA applications might require further DNA removal. Following steps provide on-membrane DNase I digestion:( see DNase I cat.# E1091for detail information) 1) For each HiBind? RNA column, prepare the DNase I digestion reaction mix as follows: OBI DNase I Digestion Buffer 73.5 ul RNase-free DNase I (20 Kunitz unites/ul) 1.5 ul Total volume 75 ul Note: a. DNase I is very sensitive for physical denaturation, so do not vortex this DNase I mixture. Mix gently by inverting the tube. Prepare the fresh DNase I digestion mixture before RNA isolation. b. OBI DNase I digestion buffer is supplied with OBI RNase-free DNase set. c. Standard DNase buffers are not compatible with on-membrane DNase digestion. 2) Pipet 75 ul of the DNase I digestion reaction mix directly onto the surface of HiBind? RNA resin in each column. Make sure to pipet the DNase I digestion mixture directly onto the membrane. DNase I digestion will not be complete if some of the mix stick to the wall or the O-ring of the HiBind? RNA column. 3) Incubate at room temperature(25-30°C) for 15 minutes.   9.  Place column in a new 2 ml collection tube and add 400 ul RNA Wash Buffer I. (If on-membrane DNase digestion was performed in the previous step, wait at least 5 minutes before proceeding). Centrifuge as above and discard flow-through.   10.  Place column in the same 2 ml collection tube and add 500 ul RNA Wash Buffer II diluted with ethanol. Centrifuge as above and discard the flowthrough. Reuse the collection tube in step 9.   Note: RNA Wash Buffer II Concentrate must be diluted with absolute ethanol before use. Refer to label on bottle for instruction.   11.  Wash column with a second 500 ul of RNA Wash Buffer II as in step 9. Centrifuge as above and discard the flow-through. Then with the collection tube empty, centrifuge the spin cartridge at 10 000 x g for 2 min at room temperature to completely dry the HiBind matrix.   12.  Transfer the column to a clean 1.5 ml micro centrifuge tube (not supplied with kit) and elute the RNA with 30-50 ul of DEPC-treated water (supplied with kit). Make sure to add water directly onto column matrix. let it sit at room temperature for 2 minutes and centrifuge at 10 000 x g for 1 min. A second elution may be necessary if the expected yield of RNA >30 ug. Alternatively, RNA may be eluted with a greater volume of water. While additional elutions increase total RNA yield, the concentration will be lowered since more than 80% of RNA is recovered with the first elution. Pre-heating the water to 65°C before adding to column and incubating column 5 min at room temperature before centrifugation may increase yields.