User Manual FavorPrep Blood / Cultured Cell Genomic
Contents
1. for 2 minutes to elute total DNA Protocol for Cultured Cell DNA Extraction Please Read Important Notes Before Starting The Following Steps 1 Transfer up to 1 x 10 of cells to a 50 ml centrifuge tube not provided Centrifuge at 4 000 x g for 5 minutes to pellet the cells If using adherent cells irypsinize the cells before harvesting 2 Resuspend the cells with 10 ml of PBS 3 Add 500 ul of Proteinase K 10 mg ml to the sample mix well by vortexing 4 Add 10 ml of FABG Buffer to the sample mixture Mix thoroughly by pulse vortexing Do not add Proteinase K directly to FABG Buffer 5 Incubate the sample mixture at 70 C for 20 minutes to lyse the sample During incubation invert the tube every 3 5 minutes At this time preheat required Elution Buffer or ddH2O 1ml per sample to 70 C For DNA Elution 6 Follow the Blood protocol starting from step 4 Troubleshooting Low yield e Too many cells were used reduce the sample volume e Poor cell lysis because of insufficient Proteinase K activity Use a fresh or well stored Proteinase K stock solution e Poor cell lysis because of insufficient mixing with FABG buffer Mix the sample and FABG Buffer immediately and thoroughly by pulse vortexing e Poor cell lysis because of insufficient incubation time Extend the incubation time and make sure that no residual particulates remain e Ethanol is not added into the lysate bef2. 0 to Proteinase K tube to make a 10 mg ml stock solution when first open Add 12 ml 32 ml of ethanol 96 100 to W1 Buffer when first open Add 80 ml 160 ml of ethanol 96 100 to Wash Buffer when first open 1 Specification Sample Size up to 10 ml of fresh frozen blood upto 1x10 of cultured cells Column Capacity up to 500 ug of Genomic DNA Average DNA yield 35 ug 1 ml whole blood Format spin column Handling Time about 1 hour Elution Volume 1 2 ml Important Notes 1 Buffers provided in this kit contain irritants Wear gloves and lab coat when handling these buffers 2 Add required volume of sterile ddH20 to Proteinase K tube to make a 10 mg ml stock solution Vortex and make sure that Proteinase K has been completely dissolved Store the stock solution at 4 C 3 Add required volume of ethanol 96 100 as Kit Contents indicated to W1 Buffer and Wash Buffer when first open 4 Use a centrifuge with a swinging bucket rotor for 50ml in all centrifugation steps The maximum speed should be 3500 5000 rpm or 3000 5000 x g 5 Preheat a dry bath or water bath to 60 C before the operation Brief Procedure Lysis FABG Buffer Proteinase K i 70 C 20minutes trif DNA Binding centmtuge Washing W1 Buffer Wash Buffer 0 DNA Elution Elution Buffer centrifuge Pure DNA co Car General Protocol for Blood DNA Extraction Please Read Important Not
3. FavorPrep Blood Cultured Cell Genomic DNA Extraction Maxi Kit User Manual Cat No FABGK 003 10 Preps FABGK 003 1 24 Preps For Research Use Only v 1005 Introduction FavorPrep Genomic DNA Extraction Maxi Kit is an excellent tool offering a speedy and economic method to purify total DNA e g genomic mitochondrial and viral DNA from whole blood fresh or frozen plasma serum buffy coat body fluids lymphocytes and cultured cells This technology first lyses cells and degrades protein by using a chaoitropic salt and Proteinase K then binds DNA to silica based membranes washes DNA with ethanol contained Wash Buffer and then elutes purified DNA by low salt Elution Buffer or ddH20 Compare with other harmful and time consuming procedures such as phenol chloroform extraction and ethanol precipitation FavorPrep shortens the handling time about 1 hour The size of purified DNA is up to 50 Kb predominantly 20 30 Kb After using FavorPrep Genomic DNA Extraction Maxi Kit the high quality total DNA can be used directly for the downstream applications Kit Contents Cat No FABGK 003 FABGK 003 1 preps 10 Preps 24 Preps Proteinase K powder 55 mg 130 mg FABG Buffer 110 ml 270 mi W1 Buffer concentrated 33 ml 88 ml Wash Buffer concentrated 20 ml 40 ml Elution Buffer 30 ml 60 ml FABG Maxi Column 10 pcs 24 pcs Elution Tube 50 ml tube 10 pcs 24 pcs User Manual 1 1 Add 5 5 ml 13 ml of sterile ddH2
4. er ethanol added to the FABG Maxi Column Close the cap and centrifuge at 4 000 x g for 5 min Discard the flow through and place the FABG Maxi Column back in the 50 ml centrifuge tube Make sure that ethanol has been added into W1 Buffer when first open 9 Add 7 ml of Wash Buffer ethanol added to the FABG Maxi Column Close the cap and centrifuge at 4 000 x g for 5 min Discard the flow through and place the FABG Maxi Column back in the 50 ml centrifuge tube Make sure that ethanol has been added into Wash Buffer when first open 10 Centrifuge at 4 000 x g for an additional 10 min to dry the column lt might be necessary to dry the column further by placing the column in a vacuum oven at 70 C for 10 minutes lmportant Step The residual liquid can affect the quality of DNA and inhibit subsequent enzymatic reactions 11 Place the FABG Maxi Column into a new 50 ml centrifuge tube Elution Tube provided 12 Add 1 ml of preheat Elution Buffer or ddH20 pH 7 5 9 0 to the membrane center of the FABG Maxi Column Stand the FABG Maxi Column for 5 min at room temperature Important Step For effective elution stand the FABG Maxi Column for 5 minutes is required to make sure that Elution Buffer is absorbed completely by column membrane Standard volume for elution is 1 ml If higher DNA yield is required repeat the DNA Elution step step 12 to increase DNA recovery 13 Centrifuge at 4 000 x g
5. es Before Starting The Following Steps 1 Transfer up to 10 ml sample whole blood buffy coat to a 50 ml centrifuge tube not provided 8 For lymphocytes sample transfer 10 10 cells to a 50 ml centrifuge tube and make total volume to 5 ml with PBS 2 Add 500 ul of Proteinase K 10 mg ml to the sample mix well by vortexing And then add 10 ml of FABG Buffer to the sample mixture Mix thoroughly by pulse vortexing Do not add Proteinase K directly to FABG Buffer 3 Incubate the sample mixture in a 60 C for 20 min to lyse the sample During incubation invert the tube every 3 5 minutes At this time preheat required Elution Buffer or ddH2O 1 2 ml per sample to 60 C For DNA Elution step 4 Optional If RNA free genomic DNA is required add 10 ul of 100 mg ml RNase A not provided to the sample mixture and incubate at room temperature for 10 minutes 5 Add 10 ml of Ethanol to the sample mixture Mix thoroughly by vortexing If precipitate appears break it by pipetting 6 Place a FABG Maxi Column to a 50 ml centrifuge tube not provided And transfer 15 ml of sample mixture ethanol added including any precipitate carefully to the FABG Maxi Column Close the cap and centrifuge at 4 000 x g for 5 min 7 Discard the flow through and transfer the rest sample mixture to the same FABG Maxi Column Close the cap and centrifuge at 4 000 x g for 5 min and discard the flow through 8 Add 4 ml of W1 Buff
6. ore transferring into FABG Maxi Column e Ethanol is not added into Wash Buffer when first open the volume or the percentage of ethanol is not correct before adding into Wash Buffer e Elution of genomic DNA is not efficient Make sure the pH of ddH20O is between 7 5 8 5 After Elution Buffer or ddH20 is added stand the FABG Maxi Column for 5 10 min before centrifugation Column is clogged Blood sample contains clots Mix the blood sample well with anti coagulant to prevent formation of blood clots e Sample is too viscous Reduce the sample volume Purified DNA dose not perform well in downstream application e Sample is old Always use fresh or well stored sample for genomic DNA extraction e Residual ethanol contamination After Wash step centrifuge at 4 000 x g for an additional 10 minutes to dry the FABG Maxi Column e RNA contamination
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