FavorPrep Tissue Total RNA Mini Kit
Contents
1. Following Steps Additional equipment xylene amp ethanol 96 100 liquid nitrogen amp mortar a rotor stator homogenizer or a 20 G needle syringe B Mercaptoethanol 70 RNase free ethanol 1 Transfer up to 15 mg paraffin embedded tissue sample to a microcentrifuge tube not provided Remove the extra paraffin to minimize the size of the sample slice Add 0 5 ml xylene mix well and incubate at room temperature for 10 min Centrifuge at full soeed for 3 min Remove the supernatant by pipetting Add 0 25 ml xylene mix well and incubate at room temperature for 3 min Centrifuge at full soeed for 3 min Remove the supernatant by pipetting Repeat step 4 and step 5 Add 0 3 ml ethanol 96 100 to the deparaffined tissue mix gently by vortexing Incubate at room temperature for 3 min 8 Centrifuge at full soeed for 3 min Remove the supernatant by pipetting 9 Repeat step 7 and step 8 10 Follow Animal tissue Protocol starting from step 1 for sample disruption then follow Animal Cells protocol starting from step 3 NOOK WN Protocol RNA clean up Please Read Important Notes Before Starting Following Steps Additional equipment xylene amp ethanol 96 100 1 Trandfer 100 ul of RNA sample to a microcentrifuge tube not provided If the RNA sample is less than 100 ul add RNase free water to make the sample volume to 100 ul 2 Add 300 ul of FARB Buffer and 300 ul of RNase free ethanol 96 100 and2. down to break down the clump 3 Place a Filter Column to a Collection Tube and transfer the sample mixture to the Filter Column Centrifuge at full soeed 18 000 x g for 2 min 4 Transfer the clarified supernatant from the Collection Tube to a new microcentrifuge tube not provided and measure the volume of the supernatant Note Avoid to pipet any debris and pellet when transfering the supernatant 5 Add 1 volume of 70 RNase free ethanol and mix well by vortexing 6 Place a FARB Mini Column to a Collection Tube and transfer the ethanol added sample mixture including any precipitate to the FARB Mini Column Centrifuge at full soeed for 1 min discard the flow through and return the FARB Mini Column back to the Collection Tube 7 Optional step DNase digestion To eliminate genomic DNA contamination follow the steps from 7a Otherwise proceed to step 8 directly 7a Add 250 ul of Wash Buffer 1 to the FARB Mini Column centrifuge at full soeed for 1 min Discard the flow through and return the FARB Mini Column back to the Collection Tube 7b Add 60 ul of RNase free DNase 1 solution 0 5U ul not provided to the membrane center of the FARB Mini Column Place the column on the benchtop for 15 min 7c Add 250 ul of Wash Buffer 1 to the FARB Mini Column centrifuge at ful soeed for 1 min Discard the flow through and return the FARB Mini Column back to the Collection Tube 7d After DNase 1 treatment proceed to step 9 8
3. Add 500 ul of Wash Buffer 1 to the FARB Mini Column centrifugeat at full soeed for 1 min Discard the flow through and return the FARB Mini Column back to the Collection Tube 9 Add 750 ul of Wash Buffer 2 to the FARB Mini Column centrifuge at full soeed for 1 min Discard the flow through and return the FARB Mini Column back to the Collection Tube Note Make sure that ethanol has been added into Wash Buffer 2 when first use 10 Repeat step 9 for one more washing 11 Centrifuge the FARB Mini Column at full soeed for an additional 3 min to dry the FARB Mini Column Important Step This step will avoid the residual liquid to inhibit subsequent enzymatic reaction 12 Place the FARB Mini Column to a Elution Tube provided 1 5 ml microcentrifuge tube 13 Add 40 100 ul of RNase free ddH20 to the membrane center of the FARB Mini Column Stand the FARB Mini Column for 1 min Important Step For effective elution make sure that RNase free ddH20O is dispensed on the membrane center and is absorbed completely Important Do not elute the RNA using RNase free water less than suggested volume lt 40 wl It will lower the RNA yield 14 Centrifuge the FARB Mini Column at full soeed for 1 min to elute RNA 15 Store RNA at 70C Protocol Isolation of Total RNA from Animal Tissues Please Read Important Notes Before Starting Following Steps Additional equipment liquid nitrogen amp mortar a rotor stator homogenizer or a 20 G needle syri
4. Following Steps Additional requirment B Mercaptoethanol 70 RNase free ethanol Enzymatic disruption Lyticase or zymolase Sorbitol buffer 1 M sorbitol 100 mM EDTA 0 1 B ME 30 C water bath or heating block Mechanical disruption 2 ml screw centrifuge tube Acid washed glass beads 500 700 um 1 Collect up to 5 x 10 of yeast culture by centrifuge at 5 000 x g for 10 min at 4 C Remove all the supernatant 2A Enzymtic disruption 2A 1 Resuspend the cell pellet in 600 ul sorbitol buffer 1 M sorbitol 100 mM EDTA 0 1 B ME not provided Add 200 U zymolase or lyticase and incubate at 30 C for 30 min Note Prepare sorbitol buffer just before use 2A 2 Centrifuge at 300 x g for 5 min to pellte the soheroplasts Remove all the supernatant 2A 3 Add 350 ul of FARB Buffer and 3 5 ul of B Mercaptoethanol to the pellet Vortex vigorously to disrupt the soheroplasts for 1 min Incbuate sample mixture at room temperature for 5 min 2B Mechanical disruption 2B 1 Add 350 ul of FARB Buffer and 3 5 ul of B Mercaptoethanol to the pellet and vortex vigorously to resuspend the cells completely 2B 2 Transfer the sample mixture to a 2 ml screw centrifuge tube and add 250 mg of acid washed glass beads 500 700 nm and vortex vigorously for 15 min to disrupt the cells 3 Follow Animal Cells Protocol starting from step 5 Protocol Isolation of Total RNA from Paraffin embedded tissue Please Read Important Notes Before Starting
5. NA Elution 4 E colli 60 RNase free water Bacteria B subtilis 1x 10 cells 40 centrifuge Yeast ee U up to 5x107 5 cerevisiae 1 x 16 cak f v 0515 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 Caution B mercaptoethanol B Me is hazardous to human health perform the procedures involving B Me in a chemical fume hood 4 Add required volume of RNase free ethanol 96 100 to Wash Buffer 2 when first use 5 All centrifuge steps are done at full soeed 18 000 x g in a microcentrifuge 6 Prepare RNase free DNase 1 reaction buffer IM NaCl 10 mM MnCl2 20 mM Tris HCI pH 7 0 at 25 C and make the final concentration of DNase to 0 5 U ul Protocol Isolation of Total RNA from Animal Cells Please Read Important Notes Before Starting Following Steps Additional requirment B Mercaptoethanol 70 RNase free ethanol _ Collect 1 5 x10 cells by centrifuge at 300 x g for 5 min at 4 C Remove all the supernatant Note Do not overload too much sample will make cell lysis incompletely and lead to lower RNA yield and purity 2 Add 350 ul of FARB Buffer and 3 5 ul of B Mercaptoethanol to the cell pellet Vortex vigorously for 1 min to resuspend the cells completely Note If the clump is still visible after vortex pipet sample mixture up and
6. User Manual FY enorer Kit Contents FavorPrep Tissue Total RNA Mini Kit Cat No FATRK 000 For isolation RNA from animal cells animal tissues bacteria FATRK 001 yeast paraffin fixed sample fungi and for RNA clean up ee ae gt For Research Use Only S a a 4 preps_sample 50 preps 100 preps 300 preps FARB Buffer 130 ml Wash Buffer 1 a 170 ml Wash Buffer 2 concentrate 50 ml x 2 RNase free Water 8 ml x 2 Filter Column 300 pcs FARB Mini Column 300 pcs Collection Tube 600 pcs Elution Tube 300 pcs Micropestle 300 pcs User Manual 1 Preparation of Wash Buffer by adding ethanol 96 100 Ethanol volume for Wash Buffer 2 6 ml 60 ml Specification Brief procedure Principle mini spin column silica matrix J Operation time 30 60 minutes Tissue Sample Binding capacity up to 100 ug total RNA column T a Column applicability centrifugation and vaccum Minimum elution volume 40 ul Cultured cells Concentration amp V Resuspension a Sample amount and yield y Lysis FARB Buffer l centrifuge j Filtration Add 70 ethanol centrifuge F RNA Binding FARB Mini Column V optional step DNase digestion Recommended amount Yield of sample used ug NIH 3T3 10 Animal cells HeLa 15 up to5x10 COS 7 1x10 cells 30 LMH 12 Sample Mouse raft Up to 30 mg Washing centrifuge Wash Buffer 1 Wash Buffer 2 x 2 4 6 Animal Tissue 9 R
7. mix well by vortexing 3 Place a FARB Mini Column to a Collection Tube and transfer the ethanol added sample mixture to the FARB Mini Column Centrifuge at full soeed for 1 min and discard the flow through and return the FARB Mini Column back to the Collection Tube 4 Follow Animal Cells Protocol starting from step 8
8. nge B Mercaptoethanol 70 RNase free ethanol A 1 Weight up to 30 mg of tissue sample Grind the sample in liquid nitrogen to a fine powder with a mortar and transfer the powder to a new microcentrifuge tube not provided Note Avoid thawing the sample during weighing and grinding A 2 Add 350 ul of FARB Buffer and 3 5 ul of B Mercaptoethanol Homogenize the sample by using a rotor stator homogenizer or by passing the sample lysate through a 20 G needle syringe 10 times Incubate at room temperature for 5 min Important step In order to release more RNA from the harder samples it is recommended to homogenize the sample by using suitable homogenize equipment for example with a rotot stator homogenizer A 3 Follow the Animal Cells Protocol starting from step 3 Alternative B 1 Place up to 30 mg of tissue sample to a microcentrifuge tube Add 350 ul of FARB Buffer and 3 5 ul of B Mercaptoethanol and use provided micropestle to grind the tissue sample thoroughly B 2 Homogenize the sample by passing lysate through a 20 G needle syringe 10 20 times Incubate at room temperature for 5 min For the tissue samples having low cell amount and hard to disrupt it is recommended to proceed A1 A3 step above B 3 Follow Animal Cells Protocol starting from step 3 Protocol Isolation of Total RNA from Bacteria Please Read Important Notes Before Starting Following Steps Additional requirment B Mercaptoethanol 70 RNase free etha
9. nol 30 C water bath or heating block 2 ml screw centrifuge tube Lysozyme reaction solution 10mg ml lysozyme 20MM Tris HCI pH 8 0 2mM EDTA 1 2 Trition Acid washed glass beads 500 700 um Transfer up to 1x1 0 cells well grown bacterial culture to a 2 ml screw centrifuge tube Note Make sure the amount of tot l RNA harvested from sample do not excess the column s binding capacity 100 ug when estimate the sample size Too much sample will make cell lysis incompletely and lead to lower RNA yield and purity If RNA amount is hard to determin on some species using lt 5x 10 cells as the starting sample size 2 Descend the bacterial cells by centrifuge at full soeed 18 000 x g for 2 min at 4 C Remove all the supernatant 3 Add 100 ul of lysozyme reaction solution Pipet up and down to resuspend the cell pellet and incubate at 37 C for 10 min 4 Add 350 ul of FARB Buffer and 3 5 ul of B Mercaptoethanol 5 Add 250 mg of acid washed glass beads 500 700 nm and vortex vigorously for 5 min to disrupt the cells 6 Centrifuge at full soeed 18 000 x g for 2 min to spin down insoluble material Transfer the supernatant to a microcentrifge tube not provided and measure the volume of the clear lysate Note Avoid pipetting any debris and pellet in the Collection Tube 7 Follow Animal Cells Protocol starting from step 5 Protocol Isolation of Total RNA from Yeast Please Read Important Notes Before Starting
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