User Manual FavorPrep Tissue Total RNA Mini Kit
Contents
1. Discard the supernatant 5 Add 350 ul of FARB Buffer B ME added to the pellet and vortex vigorously to lyse the spheroplasts Incbuate at room temperature for 5 min 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 6 Centrifuge at full speed 14 000 rpm or 10 000 x g for 2 min to spin down insoluble materials and transfer the supernatant to a microcentrifuge tube not provided 7 Follow the Animal Cells Protocol starting from step 5 6 2 Bae FavorPrep Tissue Total RNA Maxi Kit User Manual Cat No FATRK 003 10 Preps FATRK 003 1 24 Preps For Research Use Only Australian distributors v 1005 Fisher Biotec Australia free call 1800 066 077 inna lata email info fisherbiotec com GGS web www fisherbiotec com Introduction FavorPrep Tissue Total RNA Extraction Maxi Kit is desigened for extraction of total RNA from a variety of animal tissues and cells Some specially modified protocols are developed for other samples such as bacteria and yeast This method first lyses cells by using a chaotropic salt then binds RNA to silica based membranes washes RNA with ethanol contained wash buffer and then elutes purified RNA by RNase free ddH2O It takes 60 min for an entire procedure and the purified RNA is ready for RT PCR northern blotting prim2. 1 30 ml 60 ml 170 ml Wash Buffer 2 concentrated 15 mi 35 ml 50 ml x 2 RNase free ddH2O 6 ml 6 ml 8 ml X2 Filter Column 50 pcs 100 pcs 300 pcs FARB Mini Column 50 pcs 100 pcs 300 pcs Collection Tube 100 pcs 200 pcs 600 pcs Micropestle 50 pcs 100 pcs 300 pcs Elution Tube 50 pcs 100 pcs 300 pcs User manual 1 1 1 Add 60 ml ethanol 96 100 to Wash Buffer 2 when first open Add 140 ml ethanol 96 100 to Wash Buffer 2 when first open Add 200 ml ethanol 96 100 to each 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 hese buffers 3 Pipet a required volume of FARB Buffer to another RNase free container and add 10 ul B mercaptoethanol B ME per Imi FARB Buffer before use 4 Add required volume of RNase free ethanol 96 100 to Wash Buffer 2 as bottle indicated when first open 5 All centrifuge steps are done at full speed 14 000 rpm or 10 000 x g ina microcentrifuge 6 Dilute RNase free DNase 1 in reaction buffer IM NaCl 10mM MnCl2 20 mM Tris HCI pH 7 0 at 25 C to final conc 0 5 U pl 7 The additional equipment 20 G needle syringe is needed for extraction of total RNA from tissue sample Brief Procedure Cultured cells Concentration amp Resuspension Tissue Sample Homogenization Cell lysis FARB Buffer Filtration cen
3. 2 x 2 FAVORGEN BIOTECH COF FavorPrep Tissue Total RNA Mini Kit User Manual Cat No FATRK 001 50 Preps FATRK 001 1 100 Preps FATRK 001 2 300 Preps For Research Use Only Australian distributors v 1304 Fisher Biotec Australia free call 1800 066 077 iH SSE email info fisherbiotec com ae web www fisherbiotec com Introduction FavorPrep Tissue Total RNA Extraction Mini Kit is desigened for extrac tion of total RNA from animal tissue and cultured cells Some specially modified protocols are developed for other samples such as bacteria and yeast This method first lyses cells by using a chao tropic salt then binds RNA to silica based membranes washes RNA with ethanol contained wash buffer and then elutes purified RNA by RNase free ddH2O It takes 30 min for an entire procedure and the pyrified RNA is ready for RT PCR northern blotting primer extension and cDNA library contruction Sample amount and yield Recommended amount of sample used Yield ug Animal cells NIH 3T3 1 x 10 cells up to 5 x 109 HeLa 1 x 10 cells COS 7 1 x 10 cells LMH 1 x 10 cells Animal tissues Mouse rat up to 30 mg Bacteria E coli 1 x 10 cells B subtilis 1x 10 cells Yeast S cerevisiae 1 x 10 cells up to5x10 Handling time about 30 min Kit Contents Cat No FATRKOO1 FATRKOO1 1 FATRKOO1 2 preps 50 preps 100 preps 300 preps FARB Buffer 25 ml 45 ml 130 ml Wash Buffer
4. 50 ml tube not provided and adjust the volume of the clear lysate Avoid to disrupt any debris and pellet when transfer the supernatant Add an equal volume of 70 ethanol to the clear lysate and mix well by vortexing Place a FARB Maxi Column in a clean 50 ml tube not provided and transfer 14 ml of the ethanol added sample including any precipitate to FARB Maxi Colum Centrifuge at full speed for 5 min Discard the flow through and place the FARB Maxi Column back in 50 ml centrifuge Tube Then repeat this step for the rest sample mixture 8 Optional To eliminate genomic DNA contamination follow the steps from 8a Otherwise proceed to step 9 directly 8a Add 7 ml of Wash Buffer 1 to wash FARB Maxi Column centrifuge at full speed for 2 min Discard the flow through and place the FARB Maxi Column back in 50 ml centrifuge Tube 8b Add 0 5 ml of RNase free DNase 1 solution 2U ul not provided to the membrane center of FARB Maxi Column Place the Column on the benchtop for 10 min 8c Add 7 ml of Wash Buffer 1 to wash FARB Maxi Column centrifuge at full speed for 2 min Discard the flow through and place the FARB Maxi Column back in 50 ml centrifuge Tube 8d After DNase 1 treatment proceed to step 10 9 Add 12 5 ml of Wash Buffer 1 to wash FARB Maxi Column centrifuge at full speed for 2 min Discard the flow through and place the FARB Maxi Column back in 50 ml centrifuge Tube 10 Wash FARB Maxi Column twi
5. ce with 12 5 ml of Wash Buffer 2 by centrifuging at full speed for 2 min Discard the flow through and place the FARB Maxi Column back in 50 ml centrifuge Tube Make sure that ethanol has been added into Wash Buffer 2 when first open 11 Centrifuge at full speed gt 4 000 x g 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 12 Place FARB Maxi Column in Elution Tube 50 ml tube provided 13 Add 500 1000 ul of RNase free Water to the membrane center of FARB Maxi Column Stand FARB Maxi Column for 5 min Important Step For effective elution make sure that RNase free Water is dispensed on the membrane center and is absorbed completely 14 Centrifuge at full speed for 5 min to elute RNA 15 Store RNA at 70 C Special Protocol For Animal Cells 8 1 Pellet Up to 5 x10 of animal cells by centrifuge at 300 x g for 5 min Discard the supernatant completely 2 Add 14 ml of FARB Buffer B ME added to the cell pellet and vortex vigorously Incubate at room temperature for 5 min For preparation of FARB Buffer B ME added see Important Note 3 3 Place a Filter Maxi Column in a 50 ml tube not provided and transfer the sample mixture to Filter Maxi Column centrifuge at full speed for 5 min 4 Transfer the clarified supernatant from previous step to a clean 50 ml tube not provided and adjust the volume of t
6. d completely 13 Centrifuge at full speed 14 000 rpm or 10 000 x g for 2 min to elute RNA 14 Store RNA at 70C Special Protocol For Animal Tissue Additional equipment a 20 G needle syringe 1 For Fresh sample Cut up to 30 mg of tissue sample Grind the tissue sample completely in liquid nitrogen Transfer the powder to a new microcentrifuge tube not provided Or you can place tissue sample into a microcentrifuge tube and use provided micropesile to grind the tissue sample few times and break it into small pieces For Frozen sample Weight up to 30 mg tissue sample and grind the tissue sample in liquid nitrogen then transfer the powder to a new microcentrifuge tube not provided 2 Add 350 ul of FARB Buffer B ME added to the sample and shear this tissue sample by passing lysate through a 20 G needle syringe 10 times Incubate at room temperature for 5 min Grind the sample a few times to make it break more completely 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 Follow the Animal Cells Protocol starting from step 3 Special Protocol For Bacteria 1 Transfer 1 ml well grown bacterial culture or up to 1x10 cells toa microcentrifuge tube not provided 2 Descend the bacterial cells by centrifuge at full speed 14 000 rpm or 10 000 x g
7. entration amp Homogenized sample Resuspension N 4 Lysis FARB Buffeer Filtration centrifuge Total RNA Binding centrifuge Washing Wash1 Wash2 centrifuge q Total RNA Elution RNase free Water centrifuge q s CT ATI Ca Coa 1 Genernal Protocol For Animal Tissue Please Read Important Notes Before Starting The Following Steps Additional equipment a 20 G needle syringe 1 gt Cut off 0 5 g up to 1 g of tissue sample and grind the tissue sample completely under liquid nitrogen to a find powder then transfer the powder to a 50 ml centrifuge tube Note Do not use to much sample in this RNA extraction procedure It is important to use the correct number of starting cells in order to obtain optimal RNA yield and purity Add 14 ml of FARB Buffer 8 ME added to the sample and shear this tissue sample by passing lysate through a 20 G needle syringe 10 times 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 Incubate the sample mixture at room temperture for 5 minutes Place a Filter Maxi Column in a clean 50 ml tube not provided and transfer the sample mixture to Filter Maxi Column cenirifuge at full speed for 5 min Transfer the clarified supernatant from previous step to a clean
8. er extension and cDNA library contruction Sample amount 0 5 1 g of animal tissue 10 Up to5X10 bacteria cells 9 Up to 5 X 10 yeast culture 8 Up to 5 x10 of animal cells Handling time about 60 min Kit Contents Cat No preps FARB Buffer Wash Buffer 1 Wash Buffer 2 concentrated RNase free ddH2O Filter Column FARB Maxi Column Elution Tube 50 ml tube User manual FATRKOO3 10 preps 150 ml 135 ml 27 mi X2 12 ml 10 pcs 10 pcs 10 pcs 1 FATRKOO3 1 24 preps 180 ml X2 160 ml X2 27 mi X5 30 ml 24 pcs 24 pcs 24 pcs 1 Add 108 ml ethanol 96 100 to each Wash Buffer 2 when first open Important notes 1 2 3 Hi Make sure everything is RNase free when handling RNA Buffers provided in this system contain irritants Wear gloves and lab coat when handling hese buffers Pipet a required volume of FARB Buffer to another RNase free container and add 10 ul B mercaptoethanol B ME per 1ml FARB Buffer before use Add required volume of RNase free ethanol 96 100 to Wash Buffer 2 as boitle indicated when first open Dilute RNase free DNase 1 in reaction buffer 150mM NaCl 1 mM MgCl2 10 mM Tris HCI pH 7 5 to final conc 2KU ml 1 ml preparation Use a centrifuge with a swinging bucket rotor for 15ml Midi or 50ml Maxi in all centrifugation steps The maximum speed should be 3500 5000 rpm or 3000 5000 x g Brief Procedure aa a Cultured cells Conc
9. for 2 min and discard the supernatant completely 3 Resuspend the cell pellet in 100 jul of RNase free lysozyme reaction solution 20mg ml lysozyme 20mM Tris HCI pH 8 0 2mM EDTA 1 2 Trition not provided 4 Incubate at 37 C for 10 min 5 Add 350 ul of FARB Buffer B ME added to the sample and mix well by vortex Incbuate at room temperature for 5 min 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 6 Centrifuge at full speed 14 000 rpm or 10 000 x g for 2 min to spin down insouble material and transfer the supernatant to a microcentrifge tube not provided and adjust the volume of the clear lysate Avoid pipetting any debris and pellet in the Collection Tube 7 Follow the Animal Cells Protocol starting from step 5 Special Protocol For Yeast 1 Transfer 3 ml of log phase OD600 10 yeast culture to a microcentrifuge tube not provided 2 Descend the yeast cells by centrifug at 7 500 rpm 5 000 x g for 10 min and discard the supernatant completely 3 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 Prepare sorbitol buffer just before use 4 Centrifuge at 7 500 rpm 5 000xg for 5 min to pellte the spheroplasts
10. he clear lysate Avoid pipetting any debris and pellet from this Collection Tube 5 Add an equal volume of 70 ethanol to the clear lysate and mix well by pipetting 6 Follow the General Protocol starting from step 7 Special Protocol For Bacteria 10 1 Transfer Up to 5 X 10 of well grown bacterial to a enirifuge tube not provided 2 Descend the bacterial cells by centrifuge at gt 3 000 x g for 5 min and discard the supernatant completely 3 Resuspend the cell pellet in 1 ml of RNase free lysozyme reaction solution 20mg ml lysozyme 20mM Tris HCl pH 8 0 2mM EDTA 1 2 Trition not provided 4 Incubate at 37 C for 10 min 5 Add 14 ml of FARB Buffer B ME added to the sample and mix well by vortex Incubate at room temperature for 5 min For preparation of FARB Buffer B ME added see Important Note 3 6 Centrifuge at full speed for 5 min to spin down insouble material and transfer the supernatant to a 50 ml tube not provided 7 Add an equal volume of 70 ethanol to the clear lysate and mix by pipetting 8 Follow the General Protocol starting from step 7 5 Special Protocol For Yeast 1 Transfer up to 5 X 10 ml of log phase OD600 10 yeast culture to a 50 ml centrifuge tube not provided 2 Descend the yeast cells by centrifug at 500 x g at 4 C for 5 min and discard the supernatant completely 3 Resuspend the cell pellet in 2 5 ml of enzymatic lysis buffer 20 mg ml lyticase or zym
11. ntamination follow the steps from 7a Otherwise proceed to step 8 directly 7a Add 250 ul of Wash Buffer 1 to wash FARB Mini Column Centrifuge at full speed 14 000 rpm or 10 000 x g for 1 min then discard the flow through 7b Add 60 ul of RNase free DNase 1 solution 0 5U ul not provided to the membrane center of FARB Mini Column Place the Column on the benchiop for 15 min 7c Add 250 ul of Wash Buffer 1 to wash FARB Mini Column Centrifuge at full speed 14 000 rpm or 10 000 x g for 1 min then discard the flow through 7d After DNase 1 treatment proceed to step 9 8 Add 500 ul of Wash Buffer 1 to wash FARB Mini Column Centrifugeat at full speed 14 000 rpm or 10 000 x g for 1 min then discard the flow through 4 9 Wash FARB Mini Column twice with 750 jl of Wash Buffer 2 by centrifuge at full speed 14 000 rpm or 10 000 x g for 1 min then discard the flow through Make sure that ethanol has been added into Wash Buffer 2 when first open 10 Centrifuge at full speed 14 000 rpm or 10 000 x g for an additional 3 min to dry the column Important Step This step will avoid the residual liquid to inhibit subsequent enzymatic reaction 11 Place FARB Mini Column to Elution Tube 12 Add 50 ul of RNase free ddH2O to the membrane center of FARB Mini Column Stand FARB Mini Column for 1 min Important Step For effective elution make sure that RNase free ddH20 is dispensed on the membrane center and is absorbe
12. olase 1M sorbitol 100mM EDTA 0 1 B ME not provided And incubate at 30 C for 30 min Prepare sorbitol buffer just before use 4 Centrifuge at 500 x g at room temperature for 5 min to pellet spheroplasts and discard the supernatant completely 5 Add 14 ml of FARB Buffer B ME added to the sample and mix well by vortexing Incubate at room temperature for 5 minutes 6 Centrifuge at full speed for 5 min to spin down insoluble materials and transfer the clarified supernatant to a 50 ml tube not provided 7 Add an equal volume of 70 ethanol to the clear lysate and mix by pipetting 8 Follow the General Protocol starting from step 7
13. trifuge RNA Binding centrifuge Washing Wash1 Wash2 centrifuge RNA Elution RNase free Water centrifuge General Protocol For Animal Cells Please Read Important Notes Before Starting The Following Steps 1 Pellet 1 5 x10 cells by cenirifuge at 300 x g for 5 min Remove all ihe supernatant 2 Add 350 ul of FARB Buffer B ME added to the cell pellet and vortex vigorously to lyse the cells Incbuate at room temperature for 5 min 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 Place a Filter Column into a Collection Tube and transfer the sample mixture to Filter Column centrifuge at full speed 14 000 rpm or 10 000 X g for 2 min 4 Transfer the clarified supernatant from Collection Tube to a new micro centrifuge tube not provided and adjust the volume of the clear lysate Avoid pipetting any debris and pellet from Collection Tube 5 Add 1 volume of 70 ethanol to the clear lysate and mix well by vortexing 6 Briefly spin the tube to remove drops from the inside of the lid Place a FARB Mini Column into a Collection transfer the ethanol added sample including any precipitate to FARB Mini Column Centrifuge at full speed 14 000 rpm or 10 000 x g for 1 min and discard the flow through 7 Optional To eliminate genomic DNA co
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