FavorPrep Plant Total RNA Mini Kit User Manual
Contents
1. In the majority of extractions both buffer systems should provide adequate results FAPRK 62. FavorPrep Plant Total RNA Mini Kit User Manual Cat No FAPRK 001 50 Preps FAPRK 001 1 100 Preps For Research Use Only Introduction Plant Total RNA Mini Kit is specially designed for purification of total RNA from a variety of plant tissues The method uses detergents and a chaotropic salt to lyse cell and inactivate RNase In the presence of binding buffer with chaotropic salt the total RNA in the lysate binds to glass fiber matrix in the spin column The optional DNase treatments can remove DNA residues and the contaminants are washed with an ethanol contained wash buffer Finally the purified total RNA is eluted by RNase free water The protocol does not require phenol extraction and alcohol precipitation The entire procedure can be completed in 60 minutes The purified total RNA is ready for RT RT PCR real time PCR Northern blotting ssRNA and dsRNA of 200 bp to 1000 s of bps in length are efficiently purified Quality Control The quality of Plant Total RNA Mini Kit is tested on a lot to lot basis The Kits are tested by isolation of total RNA from 25 mg young leaf Purified RNA could be quantified with spectrophotometer and checked by agarose gel Sample Amount up to 100 mg plant tissue or 1 X 10 plant cells Format spin column Operation time lt 60 min Yield up to 100 ug Elution volume 50 ul 1 FAPRK Kit Contents FARB Buffer FAPRB Buffer Wash Buffer 1 Wash Buffer 2 conc RNase fre
3. d 250 ul of Wash Buffer 1 to wash FARB Mini Column Centrifuge for 1 min then discard the flow through 8d After DNase 1 treatment proceed to step 10 Add 500 ul of Wash Buffer 1 to wash FARB Mini Column Centrifuge for 1 min then discard the flow through Wash FARB Mini Column twice with 700 ul of Wash Buffer 2 by centrifuge for 1 min then discard the flow through Make sure that ethanol has been added into Wash Buffer 2 when first open Centrifuge for an additional 3 min to dry the column Important Step This step will avoid the residual liguid to inhibit the subseguent enzymatic reactions Place FARB Mini Column to a new microcentrifuge tube not provided 5 FAPRK 13 Add 50 ul of RNase free ddH O to the membrane center of FARB Mini Column Stand FARB Mini Column for 1 min 14 Centrifuge for 2 min to elute RNA 15 Store RNA at 70 C Protocol Technical Specifications Different plant species contain a lot of different metabolites like polysaccharides polyphenolics lipids or proteins Therefore we provide two different lysis buffers for the various plant samples The standard protocol uses FARB Buffer for lysis of plant sample For most of common plant species the buffer system ensures purified DNA with high yields and a good quality Alternatively buffer FAPRB is also provided with the kit The different detergent in this lysis buffer is suitable for some plant sample with a lot of polysaccharides
4. d nitrogen to a fine powder and transfer to a new microcentrifuge tube not provided 2 Add 450 ul of FARB Buffer B ME added to the sample powder and vortex vigorously Use FAPRB Buffer B ME added if plant sample contains sticky secondary metabolites such as maize with milky endosperm or mycelia of filamentous fungi 3 Place a Filter Column into a Collection Tube And transfer the mixture to Filter Column then centrifuge for 2 min 4 Transfer the clarified supernatant from the Collection Tube to a new microcentrifuge tube not provided and adjust the volume of the clear lysate 5 Add 0 5 volume of ethanol 96 100 to the clear lysate and mix by pietting For example add 250 ul of ethanol to 500 ul of the clear lysate 6 Place a FARB Mini Column into a Collection Tube And transfer 750 ul of the ethanol added sample including any precipitate to FARB Mini Column Centrifuge for 1 min and discard the flow through 7 Repeat step 6 for rest of the sample FAPRK 4 Plant RNA Mini Extraction Protocol 8 10 11 12 Optional To eliminate genomic DNA contamination follow the steps from 8a Otherwise proceed to step 9 directly 8a Add 250 ul of Wash Buffer 1 to wash FARB Mini Column Centrifuge for 1 min then discard the flow through 8b Add 100 ul of RNase free DNase 1 solution 0 5 U ul not provided to the membrane center of FARB Mini Column Place the Column on the benchtop for 15 min 8c Ad
5. e water Filter column FARB Mini column 2ml collection tube FAPRK001 50preps 30 ml 30 ml 25 ml 15 ml 6 ml 50pcs 50pcs 100pcs FAPRK001 1 100preps 60 ml 60 ml 60 ml 35 ml 6 ml 100pcs 100pcs 200pcs For FAPRK001 add 60 ml ethanol 96 100 to Wash Buffer 2 For FAPRK001 1 add 140 ml ethanol 96 100 to Wash Buffer 2 Caution The component contains irritant agent During operation always wear a lab coat disposable gloves and protective goggles References 1 Vogelstein B and Gillespie D 1979 Proc Natl Acad Sci USA 76 615 FAPRK 2 Important notes 1 Make sure everything is RNase free when handling RNA 2 Buffers provided in this system contain irritants Wear gloves and lab coat when handling these buffers 3 Pipet a required volume of FARB Buffer or FAPRB Buffer to another RNase free container and add 10 ul B mercaptoethanol B ME per 1 ml FARB Buffer or FAPRB Buffer before use 4 Add 60 ml FAPRK001 140 ml FAPRK001 1 ethanol 96 100 to Wash Buffer 2 when first open 5 All centrifuge steps are done at full speed 14 000 rpm or 10 000 xg in a microcentrifuge 6 Dilute RNase free DNase 1 in reaction buffer 1 M NaCl 10 mM MnCl 20 mM Tris HCl pH 7 0 at 25 C to final conc 0 5 U ul 3 FAPRK Plant RNA Mini Extraction Protocol Please Read Important Notes Before Starting The Following Steps 1 Grind up to 100 mg plant sample under liqui
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