User Manual FavorPrep Blood / Cultrued Cell Total
Contents
1. 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 13b Add 1 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 13c 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 cenirifuge Tube 13d After DNase 1 treatment proceed to step 15 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 Wash FARB Maxi Column twice with 12 5 ml of Wash Buffer 2 by Centrifuge 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 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 Place FARB Maxi Column to Elution Tube 50 ml tube provided 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 c2. the membrane center of FARB Mini Column Stand FARB Mini Column for 1 min Important Step For effective elution make sure that RNase free Water is dispensed on the membrane center and is absorbed completely Centrifuge at full speed 14 000 rpm or 10 000 x g for 2 min to elute RNA Store RNA at 70 C Special Protocol For Animal Cells 1 5 Pellet 1 5 x10 cells by centrifuge at 300 x g for 5 min Remove all the supernatant Add 350 ul of FARB Buffer 8 ME added to the cell pellet and vortex vigorously Incubate at room temperature for 5 min For preparation of FARB Buffer 8B ME added see Important Note 3 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 Transfer the mixture to Filter Column Set and centrifuge at full speed 14 000 rpm or 10 000 x g for 2 min Transfer the clarified supernatant from the Collection Tube to a new microcentrifuge tube not provided and adjust the volume of the clear lysate Avoid pipetting any debris and pellet from this Collection Tube Add 1 volume of 70 ethanol to the clear lysate and mix well by pipetting 6 Follow the General Protocol starting from step 12 Special Protocol For Bacteria 1 Transfer 1 ml well grown bacterial culture or up to 1x10 cells toa microcentrifuge tube not p
3. with ethanol contained wash buffer and then elutes purified RNA by RNase free Water It takes 60 min for an entire proce dure and the purified RNA is ready for RT PCR northern blotting primer extension and cDNA library contruction Sample amount 3 10 ml of human whole blood Up to 5 X 10 bacteria cells Up to 5 X 10 yeast culture Up to 5 x10 of animal cells Handling time about 60 min Kit Contents Cat No FABRK003 FABRK003 preps 10 preps 24 preps 10 X RL Buffer 100 mi 100 mi x 2 FARB Buffer 150 mi 180 mi x 2 Wash Buffer 1 135 ml 160 mi x 2 Wash Buffer 2 concentrated 27 mi x 2 27 ml x 5 RNase free Water 12 ml 30 ml Filter Column 10 pcs 24 pcs FARB Maxi Column 10 pcs 24 pcs Elution Tube 50 ml tube 10 pcs 24 pcs User manual 1 1 Add 108 ml ethanol 96 100 to each Wash Buffer 2 when first open Important notes 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 bottle 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 bucke
4. B Buffer 6 ME added to the cell pellet and vortex vigorously Incubate at room temperature for 3 min to lyse cells completely For preparation of FARB Buffer B ME added See Important Note 3 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 Place a Filter Maxi Column into a clean 50 ml tube not provided and transfer the sample mixture to Filter Column centrifuge at full speed for 5 min 10 Transfer the clarified supernatant from previous step to a clean 50 ml tube not provided and adjust the volume of the clear lysate Avoid to disrupt any debris and pellet when transfer the supernatant 11 Add an equal volume of 70 ethanol to the clear lysate and mix well by vortexing 12 13 14 15 16 17 18 19 20 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 The maximum capacity of FARB Midi Column is 14 ml repeat Step 7 for the remaining sample mixture Optional To eliminate genomic DNA contamination follow the steps from 13a Otherwise proceed to step14 direcily 13a Add 7 ml of Wash Buffer
5. FavorPrep Blood Cultrued Cell Total RNA Purification Mini Kit User Manual Cat No FABRK 001 50 Preps FABRK 001 1 100 Preps FABRK 001 2 300 Preps For Research Use Only v 1304 Introduction FavorPrep Blood Cultured Cell Total RNA Extraction Mini Kit is desi gened for extraction of total RNA from whole blood 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 chaotropic salt then binds RNA to silica based membranes washes RNA with ethanol contained wash buffer and then elutes purified RNA by RNase free ddH20 It takes 30 min for an entire procedure and the purified RNA is ready for RT PCR northern blot ting primer extension and cDNA library contruction Sample amount and yield Sample Up to 0 3 ml fresh whole blood Up to 1 X 10 animal cultured cells Up to 1 X 10 bacterial cells Up to 5 X 10 yeast Handling time about 30 min Kit Contents Cat No FABRKOO1 FABRKOO1 1 FABRKOO1 2 50 preps 100 preps 300 preps preps RL Buffer 120 ml 240 mi 240 mI X 3 FARB Buffer 25 mi 45 ml 130 ml Wash Buffer 1 30 ml 60 ml 170 ml Wash Buffer 2 concentrated 15 ml 35 ml 50 ml x 2 RNase free ddH20 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 Elution Tube 50 pcs 100 pcs 300 pcs User manual 1 1 1 Add 60 ml ethan
6. cipitate to FARB Mini Column Set Centrifuge at full speed 14 000 rpm or 10 000 x g for 1 min and discard the flow through 13 Optional To eliminate genomic DNA contamination follow the steps from 13a Otherwise proceed to step14 directly 13a 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 13b 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 benchtop for 15 min 13c 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 13d After DNase 1 treatment proceed to step 15 14 Add 500 ul of Wash Buffer 1 to wash FARB Mini Column Centrifuge at full 15 16 17 18 19 20 speed 14 000 rpm or 10 000 x g for 1 min then discard the flow through Wash FARB Mini Column twice with 700 ul 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 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 Place FARB Mini Column to Elution Tube Add 50 ul of RNase free Water to
7. e tube 1 5 ml or 2 0 ml tube not provided If the sample volume is more than 200 ul use a 2 0 ml tube as the sample container Mix 5 volume of RL Buffer with 1 volume of the sample and mix well by inversion Incubate on ice for 10 min Vortex briefly 2 times during incubation Centrifuge for 1 min at 4 500 rpm to form a cell pellet and discard the supernatant completely Add 600 ul of RL Buffer to resuspend the cell pellet by briefly vortexing Centrifuge for 1min at 4 500 rpm to form a cell pellet again and discard the supernatant completely Add 350 ul of FARB Buffer G ME added to the cell pellet and vortex vigoruusly Incubate at room temperature for 5 min For preparation of FARB Buffer B ME added See Important Note 3 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 10 11 12 Transfer the sample mixture to Filter Column Set and centrifuge at full speed 14 000 rpm or 10 000 x g for 2 min Transfer the clarified supernatant from the Collection Tube to a new microcentrifuge tube not provided and adjust the volume of the clear lysate Avoid to pipette any debris and pellet from the Collection Tube Add 1 volume of 70 ethanol to the clear lysate and mix well by vortexing Transfer the ethanol added sample including any pre
8. enter and is absorbed completely Centrifuge at full speed for 5 min to elute RNA 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 8 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 the 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 Genernal Protocol starting from step 12 Special Protocol For Bacteria 10 1 Transfer Up to 5 X 10 of well grown bacterial to a centrifuge 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 HCI pH 8 0 2mM EDTA 1 2 Trition not provided 4 Incubate at 37 C for 10 min 5 Add 13 ml of FARB Buffer 8 ME added to the sample and mix well by vorte
9. epare sorbitol buffer just before use 4 Centrifuge at 7 500rpm 5 000xg for 5 min Remove the supernatant by pipetting 5 Add 350ul of FARB Buffer 8 ME added to the sample and mix well by vortexing Incubate 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 microc entrifuge tube not provided 7 Add 250 ul of ethanol 96 100 to the clear lysate and mix by pipetting 8 Follow the General Protocol starting from step 12 z E FAVORGEN FavorPrep Blood Cultured Cell Total RNA Purification Maxi Kit User Manual Cat No FABRK 003 10 Preps FABRK 003 1 24 Preps For Research Use Only v 1005 Australian distributors Fisher Biotec Australia free call 1800 066 077 Ha S email info fisherbiotec com austra i web www fisherbiotec com Introduction FavorPrep Blood Cultured Cell Total RNA Extraction Maxi Kit is desi gened for extraction of total RNA from whole blood 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 chaotropic salt then binds RNA to silica based membranes washes RNA
10. ol 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 1 Important notes 1 Make sure the starting sample amount is under the limit 2 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 bottle indicated when first open The additional equipment 20 G needle syringe is needed for extraction of total RNA from tissue sample Dilute RNase free DNase 1 in reaction buffer to final conc 0 5 U ul Brief Procedure o Whole Blood v FF Cultured cells 3 Concentration amp RL lysis RL Buffer gt y Y Resuspension J Cell lysis FARB Buffer RNA Binding centrifuge centrifuge centrifuge Washing Wash1 Wash2 gt wf v RNA Elution Genernal Protocol For Human Whole Blood Please Read Important Notes Before Starting The Following Steps 1 2 Collect fresh human blood in an anticoagulant treat collection tube Add 200 300 pl human whole blood to an appropriately sized micro centrifug
11. rovided Descend the bacterial cells by centrifuge at full speed 14 000 rpm or 10 000 x g for 2 min and discard the supernatant completely Resuspend the cell pellet in 100 ul RNase free lysozyme reaction solution 20mg ml lysozyme 20mM Tris HCI pH 8 0 2mM EDTA 1 2 Trition not provided Incubate at 37 C for 10 min Add 350 ul of FARB Buffer 8 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 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 7 Add 1 volume of 70 ethanol to the clear lysate and mix by pipetting 8 Follow the General Protocol starting from step 12 Special Protocol For Yeast 1 Transfer 3 ml 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 1M sorbitol 100mM EDTA 0 1 B ME not provided Add 200 U zymolase or lyticase and incubate at 30 C for 30 min Pr
12. t 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 Cultured cells RL lysis diulted RL Buffer gt lt Concentration amp Resuspension N Lysis FARB Buffeer j lt 0 centrifuge q Filtration i centrifuge Total RNA Binding E centrifuge Washing Wash1 Wash2 L centrifuge Total RNA Elution i RNase free Water N Genernal Protocol For Human Whole Blood Please Read Important Notes Before Starting The Following Steps 1 2 on Collect fresh human blood in an anticoagulant treat collection tube Add 3 10 ml of human whole blood to an appropriately sized centrifuge tube 15 ml or 50 ml tube not provided Mix 5 volume of diluted RL Buffer with 1 volume of the sample and mix well by inversion Note RL buffer is provided as a 10 x concentrate which would must be diluted with sterile deionized water before use For example add 25 ml of diluted RL Buffer to 5 ml of blood sample Incubate at room temperature for 5 min Vortex briefly 2 times during incubation Centrifuge for 5 min at 500 x g to form a cell pellet and discard the supernatant completely Add 2 volume of diluted RL Buffer to wash the cell pellet by briefly vortexing Centrifuge for 5 min at 500 x g to form a cell pellet again and discard the supernatant completely Add 12 5 ml of FAR
13. x 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 Genernal Protocol starting from step 12 5 Special Protocol For Yeast 1 2 Transfer up to 5 X 10 yeast cells to a 50 ml centrifuge tube not provided Descend the yeast cells by centrifug at 500 x g at 4 C for 5 min and discard the supernatant completely Resuspend the cell pellet in 2 5 ml of enzymatic lysis buffer 20 mg ml lyticase or zymolase 1M sorbitol 100mM EDTA 0 1 B ME not provided And incubate at 30 C for 30 min Prepare sorbitol buffer just before use Centrifuge at 500 x g at room temperature for 5 min to pellet spheroplasts and discard the supernatant completely Add 14 ml of FARB Buffer 8 ME added to the sample and mix well by vortexing Incubate at room temperature for 5 minutes Centrifuge at full speed for 5 min to spin down insoluble materials and transfer the clarified supernatant to a 50 ml tube not provided Add an equal volume of 70 ethanol to the clear lysate and mix by pipetting Follow the Genernal Protocol starting from step 12
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