User Manual FavorPrep Total RNA Mini Kit (Blood/Cultured Cell)
Contents
1. 100 ul of RBC Lysis Buffer Blood Cultured Cell Protocol Step 1 Cell Lysis 1 2 Add 400 ul of FARB Buffer B ME added to the sample and vortex vigorously For preparation of FARB Buffer 8 ME added See Important Note 3 Incubate at room temperature for 5 minutes Step 2 Binding 3 Add 500 ul of 70 ethanol to the sample and shake vigorously pipetting if there is any precipitate Place a RB Column in a 2 ml Collection Tube Transfer the ethanol added mixture to the RB Column Centrifuge for 1 minute at full speed 14 000 rpm or 10 000 x g and discard the flow through Repeat step 5 for any rest sample Place the RB Column in a new 2 ml Collection Tube Optional Step DNA residue degradation To eliminate genomic DNA contamination follow the optional steps Add 100 ul of DNase 2 KU ml mixed in a reaction buffer 50 mM Tris HCI pH 7 5 10 mM MnCl2 50 pg ml BSA at 25 C to the center of the FARB Column matrix Place the Column on the benchtop for 10 minutes at room temperature Blood Cultured Cell Protocol Step 3 Washing 9 10 11 Add 400 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 Wash FARB Mini Column twice with 600 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 Wash2. 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 lmportant Step This step will avoid the residual liquid to inhibit subsequent enzymatic reaction Step 4 Elution 12 13 14 15 Place FARB Mini Column to RNase free 1 5ml microcentrifuge tube Add 50 ul of RNase free Water to the membrane center of FARB Mini Column Stand FARB Mini Column for 3 min or until the water has been absorbed by the matrix 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 1 min to elute RNA Optional Step DNA residue degradation To eliminate genomic DNA contamination follow the optional steps Add 2 ul of DNase 2 KU ml mixed in a reaction buffer 50 mM Tris HCl pH 7 5 10 mM MnCl2 50 pg ml BSA at 25 C to the final elution sample Stand for 10 minutes at room temperature Step Final Pure RNA 16 Store RNA at 70 C Bacteria Protocol Sample Preparation For Gram negative bacteria i Transfer the appropriate number of cell up to 1 x 109 to a 1 5ml microcentrifuge tube not provided and centrifuge at full speed 14 000 rpm or 10 000 x g for 1 minute then discard the supernatant ii Vortex the pellet for 30 seconds iii Add 200 ul of FART Buffer and resuspend the pellet by vortex iv Incubate f
3. FavorPrep Total RNA Mini Kit Blood Cultured Cell User Manual Cat No FABRK 100 100 Preps FABRK 300 300 Preps For Research Use Only Kit Contents Cat No FABRK 100 preps 100 Preps RBC Lysis Buffer 200 ml FARB Buffer 60 ml FART Buffer 30 ml Wash Buffer 1 50 ml Wash Buffer 2 concentrated 25 ml RNase free Water 6 ml FARB Column 100 pcs 2 ml Collection Tube 200 pcs FABRK 300 300 Preps 500 ml 130 ml 75 ml 130 ml 50 mi x 2 30 ml 300 pcs 600 pcs Add 100 ml 200 ml of ethanol 96 100 to Wash Buffer when first open Blood Cultured Cell Protocol Sample Preparation For Fresh whole human blood i Collect fresh human blood in an anticoagulant ireat collection tube Add 1 ml of RBC Lysis Buffer to an appropriately sized micro centrifuge tube 1 5 ml or 2 0 ml tube not provided iii Add 300 pl of whole human blood and mix by inversion v Incubate on ice for 10 minutes Vortex briefly 2 times during incubation lt Centrifuge for for 5 minutes at 1 000 x g at 4 C to form a cell pellet and discard the supernatant completely vi Resuspend the pellet with 100 pl of RBC Lysis Buffer by pipetting For Cultured animal cells i Trypsinize the adherent cells before harvesting ii Transfer the appropriate number of cell up to 5 x 106 to a 1 5ml microcentrifuge tube not provided and centrifuge at 6000 x g for 20 seconds iii Remove the supernatant and resuspend the cells with
4. at 2 000 x g for 10 minutes to harvest the spheroplast and then remove the supernatant Follow the Bacteria Protocol starting from Step 1 Cell Lysis
5. or 5 minutes at room temperature For Gram positive bacteria i Transfer the appropriate number of cell up to 1 x 109 to a 1 5ml microcentrifuge tube not provided and centrifuge at 6000 x g for 1 minute then discard the supernatant ii Add 200 ul of lysozyme buffer 20 mg ml lysozyme 20 mM Tris HCI 2 mM EDTA 1 Triton X 100 pH 8 0 prepare fresh lysozyme buffer immediately prior to use and resuspend the pellet by vortex iii Incubate for 10 minutes at room temperature During incubation invert the tube every 2 3 minutes Step 1 Cell Lysis 1 Add 300 ul of FARB Buffer 8 ME added to the sample and mix well by vortex Incubate for 5 minutes at room temperature For preparation of FARB Buffer 8 ME added see Important Note 3 2 Centrifuge at full speed 14 000 rpm or 10 000 x g for 2 minutes to spin down insouble material and transfer the supernatant to a new microcentrifge tube not provided 3 Follow the Blood Cultured Cell Protocol starting from Step 2 Binding Fungus Protocol Sample Preparation Harvest appropriate number of cell up to 5 x 107 to a 1 5ml microcentrifuge tube not provided and centrifuge at 5000 x g for 10 minute then discard the supernatant ii Add 600 ul of sorbitol buffer 1 2 M sorbitol 10 mM CaCl2 0 1 M Tris HCI pH 7 5 35mM mercaptoethanol and resuspend the pellet iii Add 200 U of lyticase or zymolase Incubate for 30 minutes at 30 C iv Centrifuge the mixture
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