FavorPrep Plant Total RNA Maxi kit User Manual
Contents
1. sticky secondary metabolites such as maize with milky endosperm or mycelia of filamentous fungi Note In order to release all the RNA in the sample it is required to disrupt the sample completely Different samples require different methods ex disruptor equipment to achieve complete disruption 3 Incubate at 70 C for 10 min vortex every 3 min during incubation 4 Place a Filter Column to a 50 ml centrifuge tube not provided And transfer the entire sample mixture to the Filter Column 5 Centrifuge at full speed 4500 6 000 rpm for 5 min at 4 C 6 Transfer the clarified flow through to a new 50 ml centrifuge tube not provided and adjust the volume of the clarified flow through Avoid pipett any debris and pellet when transfering the clarified flow through 7 Add 1 volume of 70 ethanol to the clarified flow through and mix well by plus vortexing for 5 seconds For example add 4 5 ml of 70 ethanol to 4 5 ml of clearified flow through 8 Place a FARB Maxi Column to a 50 ml cenirifuge tube not provided Transfer the ethanol added sample mixture including any precipitate to the FARB Maxi Column Centrifuge at full speed 4500 6 000 rpm for 1 min Discard the flow through and place the FARB Maxi Column back to the 50 ml centrifuge tube 9 Optional To eliminate genomic DNA contamination of RNA follow the steps from 9a Otherwise proceed to step 10 directly 9a Add 2 5 ml of Wash 1 Buffer to the2. 24 pcs FARB Maxi Column 10 pcs 24 pcs Add 50 ml ethanol 96 100 to Wash Buffer 2 when first open Add 200 ml ethanol 96 100 to Wash Buffer 2 when first open 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 5 ml of FARB Buffer to another RNase free container and add 50 ul of 8 mercaptoethanol B ME before every preparation 4 Add required amount of ethanol 96 100 Wash 2 Buffer when first open 5 Dilute RNase free DNase 1 in reaction buffer IM NaCl 10mM MnClz 20mM Tris HCl pH7 0 at 25 C to final conc 0 5U ul Brief Procedure Homogenized plant sample under liquid nitrogen L Lysis TRX Buffeer 70 C for 10 min l centrifuge i Filtration centrifuge E Total RNA Binding centrifuge T Washing Wash1 E Wash2 centrifuge Total RNA Elution RNase free Water Genernal Protocol Please Read Important Notes Before Starting The Following Steps 1 Grind 500 mg up to 1 g of plant sample under liquid nitrogen to a fine powder and transfer to a new 50 ml centrifuge tube not provided Note Do not use plant sample more than 14g it will lower the total RNA yield 2 Add 5 ml of FARB Buffer 8 ME added to the sample powder and vortex vigorously Use FAPRB Buffer 8 ME added if plant sample contains
3. FARB Maxi Column Centrifuge at full speed 4500 6 000 rpm for 2 min Discard the flow through and place the FARB Maxi Column back to the 50 ml centrifuge tube 9b Add 800 ul of RNase free DNase 1 solution 0 5 U ul not provided to the membrane center of FARB Maxi Column Place the Column on the benchtop for 15 min 9c Add 2 5 ml of Wash 1 Buffer to the FARB Maxi Column Centrifuge at full speed 4500 6 000 rpm for 2 min Discard the flow through and place the FARB Maxi Column back to the 50 ml centrifuge tube 9d After DNase 1 treatment proceed to step 11 10 Add 5 ml of Wash 1 Buffer to wash the FARB Maxi Column Cenirifuge at full speed 4500 6 000 rpm for 2 min Discard the flow through and place the FARB Maxi Column back to the 50 ml centrifuge tube 11 Wash FARB Maxi Column twice with 5 ml of Wash 2 Buffer by Centrifuge at full speed 4500 6 000 rpm for 2 min Discard the flow through and place the FARB Maxi Column back to the 50 ml centrifuge tube Make sure that ethanol has been added into Wash 2 Buffer when first open 12 Centrifuge at full speed 4500 6 000 rpm for an additional 10 min to dry the FARB Maxi column Important Step This step will avoid the residual liquid to inhibit subsequent enzymatic reaction 13 14 15 16 Place the FARB Maxi Column to a new 50 ml centrifuge not provided Add 1 ml of RNase free Water to the membrane center of the FARB Maxi Column Stand the FARB Maxi Column
4. FavorPrep Plant Total RNA Maxi kit User Manual Cat No FAPRK 002 10 Preps FAPRK 002 1 24 Preps For Research Use Only v 1302 Introduction FavorPrep Plant Total RNA Maxi Kit provides a fast and simple method to isolate total RNA from plant tissue and cells In the process sample is homogenized by grinding the plant tissue in liquid nitrogen and filtrated by filter column to remove cell debris 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 precipita tion The entire procedure can be completed in 60 minutes The purified total RNA is ready for RT RT PCR real time PCR Northern blotting Sample amount and yield Sample Amount 500 mg up to 1 g plant tissue or 5 10 X 10 plant cells Operation time about 45 60 min Binding Capacity up to 1000 ug total RNA Expected Yield up to 50 300 pg total RNA from young leave Elution volume 500 ul Kit Contents Cat No FAPRK 002 FAPRK 002 1 preps 10 preps 24 preps FARB Buffer 60 ml 140 ml FAPRB Buffer 60 ml 140 ml Wash Buffer 1 45 ml 140 ml Wash Buffer 2 concentrated 12 5 ml 50 ml RNase free Water 6 ml 30 ml Filter Column 10 pcs
5. for 5 min Important Step For effective elution make sure that the elution solution is dispensed of the membrane center and is absorbed completely Centrifuge at full speed 4500 6 000 rpm for 5 min to elute RNA Store RNA at 70 C
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