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1. 2 Buffer for gel electrophoresis contaminated with DNase Use fresh running buffer for gel electrophoresis FABGK 12 Storage Conditions FavorPrep Genomic DNA Extraction Mini Kit except Proteinase K can be stored at room temperature 15 25 C for up to 1 year Proteinase K powder can be stored dry at room temperature for up to 6 months For storage longer than 6 months Proteinase K powder should be stored dry at 2 8 C Proteinase K stock solution is stable for 2 months when stored at 2 8 C Storage at 20 C will prolong its life but repeated freezing and thawing should be avoided 13 FABGK Q i GENOREEN FavorPrep Genomic DNA Mini Kit Blood Cultured Cell User Manual Cat No FABGK 100 100 Preps FABGK 300 300 Preps For Research Use Only Australian distributors Fisher Biotec Australia free call 1800 066 077 iHa SE email info fisherbiotec com austra J web www fisherbiotec com Kit Contents Cat No FABGK 100 preps 100 Preps RBC Lysis Buffer 135 ml FATG Buffer 30 ml FABG Buffer 40 ml W1 Buffer 45 ml Wash Buffer concentrated 25 ml Elution Buffer 30 ml FABG Column 100 pcs 2 ml Collection Tube 200 pcs FABGK 300 300 Preps 405 ml 75 ml 100 ml 130 ml 50 ml 75 ml 300 pcs 600 pcs Add 100 ml 200 ml of ethanol 96 100 to Wash Buffer when first open Specification Sample Size Up to 300 ul of Whole blood Up to 200 ul of frozen blood Up to 200 ul of buffy2. up to 50 ug of total DNA depends on the sample types and the number of cells in the sample Handling time within 1 hour depends on the sample types 1 FABGK Kit Contents FABGK001 50preps FABG Buffer 15 ml W1 Buffer 22 ml Wash Buffer 10 ml Elution Buffer 15 ml Proteinase K 11mg FABG Mini Column 50 pcs Collection Tube 100 pcs Elution Tube 50 pcs FABGKOO1 1 100preps 30 ml 44 ml 20 ml 30 ml ii mgx2 100 pcs 200 pcs 100 pcs FABGK001 2 300preps 70 ml 124 ml 50 ml 30 ml x 3 11mgx6 300 pcs 600 pcs 300 pcs Add 8 16 45 ml ethanol 96 100 to W1 Buffer when first open Add 40 80 200 ml ethanol 96 100 to Wash Buffer when first open Add 1 1 ml sterile ddH O to each Proteinase K tube to make a 10mg ml stock solution FABGK 2 Important Notes 1 Buffers provided in this system contain irritants Wear gloves and lab coat when handling these buffers 2 Add 1 1 ml sterile ddH O to each Proteinase K tube to make a 10mg ml stock solution Vortex and make sure that Proteinase K has been completely dissolved Store the stock solution at 4 C 3 For FABGK 001 50preps add 8ml ethanol 96 100 to W1 Buffer when first open For FABGK 001 1 100presp add 16 ml ethanol 96 100 to W1 Buffer when first open For FABGK 001 2 300presp add 45 ml ethanol 96 100 to W1 Buffer when first open 4 For FABGK001 50preps add 40ml ethanol 96 100 to Wash Buffer when first open
3. For FABGK 001 1 100preps add 80 ml ethanol 96 100 to Wash Buffer when first open For FABGK 001 2 300preps add 200 ml ethanol 96 100 to Wash Buffer when first open 5 Preheat a dry bath or a water bath to 60 C before the operation 6 All centrifuge steps are done at full speed 14 000 rpm or 10 000 xg in a microcentrifuge 3 FABGK Brief Procedure Cell lysis FABG Protein degradation Proteinase K k Binding centrifuge q E 2 Wash W1 Buffer Wash Buffer centrifuge q 3 Elution Elution Buffer centrifuge q 7 Pure genomic DNA FABGK 4 General Protocol Please Read Important Notes Before Starting The Following Steps HINTP Preheat a 60 C dry bath or water bath for step 4 1 Transfer up to 200 ul sample whole blood buffy coat to a micropcentrifuge tube not provided If the sample volume is less than 200 ul add the appropriate volume of PBS 2 Optional If RNA free genomic DNA is required add 4 ul of 100 mg ml RNase A to the sample and incubate for 2 minutes at room temperature 3 Add 20 ul Proteinase K and 200 pl FABG Buffer to the sample Mix thoroughly by pulse vortexing Do not add Proteinase K directly to FABG Buffer 4 Incubate at 60 C for 15 minutes to lyse the sample During incubation vortex the sample every 3 5 minutes 5 Briefly spin the tube to remove drops from the inside of the lid 6 Add 200 ul ethanol 96 100 to the sample Mi
4. coat Up to 1 x 107 of Cultured animal cells Up to 1 x 10 of Cultured bacterial cells Up to 5 x 107 of Fungus cells Average Yield about 50 ug Format spin column Handling time within 60 minutes Important Notes 1 Buffers provided in this system contain irritants Wear gloves and lab coat when handling these buffers 2 For Cat No FABGK 100 add 100 ml of ethanol 96 100 to Wash Buffer when first open For Cat No FABGK300 add 200 ml of ethanol 96 100 to Wash Buffer when first open Brief Procedure Fresh Blood v RBC Iysis Cell lysis FABG Binding centrifuge Washing W1 Buffer Wash Buffer centrifuge Elution Elution Buffer centrifuge Pure genomic DNA Genernal Protocol For Fresh Blood Please Read Important Notes Before Starting The Following Steps Step 1 RBC Lysis 1 Collect fresh human blood in an anticoagulant ireat collection tube 2 Transfer up to 300yul fresh blood to a 1 5ml microcentrifuge tube not provided If the sample is more than 300 pl up to 1 ml add the sample to a sterile 15 ml centrifuge tube 3 Add 3 x the sample volume of RBC Lysis Buffer and mix by inversion Do not vortex 4 Incubate at room temperature for 10 minutes 5 Centrifuge at 3 000 x g for 5 minutes and completely remove the supernatant 6 Resuspend the pellet with 100 pl of RBC Lysis Buffer Step 2 Cell Lysis 7 Add 200 ul of FABG Buffer a
5. first open Repeat the extraction procedure with a new sample B The volumn or the percentage of ethanol is not correct before adding into Wash Buffer Make sure that the correct volumnes of ethanol 96 100 is added into Wash Buffer when first open Repeat the extraction procedure with a new sample Column is clogged 1 Blood sample contains clots Repeat the extraction procedure with a new sample Mix the blood sample well with anticoagulant to prevent formation of blood clots 2 Sample is too viscous Reduce the sample volume 3 Insufficient activity of Proteinase K Use a fresh or well stored Proteinase K Stock solution Repeat the extraction procedure with a new sample Do not add Proteinase K Into FABG Buffer directly FABGK 10 Trouble Shooting Problem Possible Reasons Solution A s A220 ratio of eluted DNA is low Poor cells lysis 1 Poor cell lysis because of insufficient Proteinase K activity Repeat the extraction procedure with a new sample Use a fresh or well stored Proteinase K stock solution Do not add Proteinase K directly to FABG Buffer 2 Poor cell lysis because of insufficient mixing with FABG Buffer Repeat the extraction procedure with a new sample Mix the sample and FABG Buffer immediately and throughly by pulse vortexing 3 Poor cell lysis because of insufficient incubation time Repeat the extraction proce
6. min at full speed 14 000 rpm or 10 000 x g to dry the column Important Step This step will avoid the residual liquid to inhibit subsequent enzymatic reactions Step 6 Elution 16 17 18 19 Place the dry FABG Column to a new 1 5ml microcentrifuge tube Add 100ul of Preheated Elution Buffer or TE to the membrane center of FABG Column Important Step For effective elution make sure that the elution solution is dispensed onto the membrane center and absorbed completely Incubate the FAGB Column at 37 C for 10 minutes in an incubator Centrifuge for 1 minute at full speed 14 000 rpm or 10 000 x g to elute the DNA Standard volume for elution is 100 ul If higher DNA yield is required repeat the DNA Elution step to increase DNA recovery and the total volume could be 200 ul Step Final Pure DNA 20 Store the DNA fragment at 4 C or 20 C Special Protocol For Cultured Cell Step 1 Sample Preparation A For Cultured Animal Cells i Trypsinize the adherent cells before harvesting ii Transfer the appropriate number of cell up to 1 x 10 to a 1 5ml microcentrifuge tube not provided and centrifuge at 6 000 x g for 20 seconds iii Remove the supernatant and resuspend the cells with 150 ul of RBC Lysis Buffer Then follow Step 2 Cell Lysis B For Fresh blood except human blood i The sample volume of mammalian blood non nucleated can be up to 50ul the sample volume of nuc
7. 2 G FAVORGEN FavorPrep Blood Cultured Cell Genomic DNA Extraction Mini Kit User Manual Cat No FABGK 001 50 Preps FABGK 001 1 100 Preps FABGK 001 2 300 Preps For Research Use Only v 1002 Australian distributors Fisher Biotec Australia free call 1800 066 077 Introduction FavorPrep Genomic DNA Extraction Mini 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 chaotropic 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 ddH O Compare with other harmful and time consuming procedures such as phenol chloroform extraction and ethanol precipitation FavorPrep shortens the handling time within 1 hour The size of purified DNA is up to 50 Kb predominantly 20 30 Kb After using FavorPrep Genomic DNA Extraction Mini Kit the high quality total DNA can be used directly for the downstream applications Specification Sampling up to 200 ul of whole blood with anti coagulant plasma serum buffy coat or body fluids up to 5 x 10 lymphocytes or cultured cells in 200 ul PBS Yield about 5 ug of total DNA from 200 ul of human whole blood
8. Protocol For cultured Cells 1 Harvest Cells a Cells grown in suspension i Transfer the appropriate number of cell up to 5 x 10 to a 1 5ml microcentrifuge tube not provided ii Centrifuge at 300 x g for 5 minutes iii Remove the supernatant carefully and completely b Cells grown in monolayer i Detach cells from the dish or flask by trypsinization or using a cell scraper ii Transfer the appropriate number of cell up to 5 x 10 to a 1 5ml microcentrifuge tube not provided iii Centrifuge at 300 x g for 5 minutes iv Remove the supernatant carefully and completely 2 Resuspend cell pellet in PBS to a final volume of 200ul 3 Follow the General Protocol starting from step 2 Preparation of buffy coat Centrifuge whole blood at 3 300xg for 10 minutes at room temperature and you will get three different fractions the upper clear layer is plasma the intermediate layer is buffy coat containing concentrated leukocytes the bottom layer contains concentrated erythrocytes Process the General Protocol from Step 1 for buffy coat Extraction total DNA from buffy coat will yield 5 10 times more DNA than an equivalent volume of whole blood 7 FABGK Trouble Shooting Problem Possible Reasons Solution Low or no yield of genomic DNA 1 Low amount of cells in the sample Concentrate a larger volume of a new sample to 200 ul If the sample is whole blood prepare buffy coat refer to Special Protocol on p
9. age 7 2 Poor cell lysis A it is because of insufficient Proteinase K activity Repeat the extraction procedure with a new sample Use a fresh or well stored proteinase K stock solution B it is because of insufficient mixing with FABG Buffer Repeat the extraction procedure with a new sample Mix the sample and FABG Buffer immediately and thoroughly by pulse vortexing C It is because of insufficient incubation time Repeat the extraction procedure with a new sample Extend the incubation time and make sure that no residual particulates remain 3 Ethanol is not added into the lysate before transferring sample mixture into FABG Column Repeat the extraction procedure with a new sample 4 Incorrect preparation of Wash Buffer A Ethanol is not added into Wash Buffer when first open Make sure that the correct volumes of ethanol 96 100 is added into Wash Buffer when first open Repeat the extraction procedure with a new sample FABGK 8 Trouble Shooting Problem Possible Reasons Solution Low or no yield of genomic DNA A B The volume or the percentage of ethanol is not correct before adding into Wash Buffer Make sure that the correct volumes of ethanol 96 100 is added into Wash Buffer when first open Repeat the extraction procedure with a new sample Elution of genomic DNA is not efficient PH of water ddH O for elution is acidic Make sure th
10. arred when processing binding step Step 3 Cell Lysis 7 Add 250 ul of FABG Buffer to the sample and mix by vortex 8 Incubate for 30 minutes at room temperature or until the sample lysate is clear During incubation invert the tube every 3 minutes 9 Preheat required Elution Buffer for Step 6 DNA Elution in a 70 C water bath 10 Optional Step If RNA free genomic DNA is required add 5ul of 10 mg ml RNase A to the sample and mix by vortex Then incubate for 5 minutes at room temperature Step 4 Binding 11 12 Add 250 1 ethanol 96 100 to the sample and vortex for 10 seconds Pipetting if there is any precipitate Place a FABG Column to a 2ml collection tube Transfer the sample mixture including any precipitate to FABG Column Cenirifuge for 5 minute at full speed 14 000 rpm or 10 000 x g and discard the 2ml collection tube Place the FABG Column in a new 2ml Collection tube Step 5 Washing 13 14 15 Wash FABG Column with 400ul W1 Buffer Centrifuge for 1 min at full speed 14 000 rpm or 10 000 x g and discard the flow through Place the FABG Column back in the 2ml Collection tube Wash FABG Column with 600ul Wash Buffer ethanol added Centrifuge for 1 min at full speed 14 000 rpm or 10 000 x g and discard the flow through Make sure that ethanol has been added into Wash Buffer when first open Place the FABG Column back in the 2ml Collection tube Centrifuge for an additional 3
11. at required Elution Buffer for Step 5 DNA Elution in a 70 C water bath 6 Optional Step If RNA free genomic DNA is required add 5ul of 10 mg ml RNase A to the sample and mix by vortex Then incubate for 5 minutes at room temperature 7 Follow the General Protocol starting from Step 3 Binding Special Protocol For Buffy Coat Step 1 Sample Preparation Centrifuge whole blood at 3 300xg for 10 minutes at room temperature and you will get three different fractions the upper clear layer is plasma the intermediate layer is buffy coat containing concentrated leukocytes the bottom layer contains concentrated erythrocytes Extraction total DNA from buffy coat will yield 5 10 times more DNA than an equivalent volume of whole blood Step 2 RBC Lysis 1 Transfer up to 200ul buffy coat to a 1 5ml microcentrifuge tube not provided 2 Add 3 x the sample volume of RBC Lysis Buffer and mix by inversion 3 Incubate at room temperature for 10 minutes During incubation invert the tube every 3 minutes 4 Centrifuge for 1 minutes at full speed 14 000 rpm or 10 000 x g and completely remove the supernatant 5 Resuspend the pellet with 500 pl of RBC Lysis Buffer Then centrifuge for 1 minutes at full speed 14 000 rpm or 10 000 x g and completely remove the supernatant 6 Resuspend the pellet with 200 ul of RBC Lysis Buffer Mix the tube by vortex only Be sure the pellet is completely resuspended or the column would be b
12. ause 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 before transferring into FABG Midi 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 ddH20 is between 7 5 8 5 After Elution Buffer or ddH20 is added stand the FABG Midi 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 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 Midi Column e RNA contamination
13. dure with a new sample Extend the incubation time and make sure that no residual particulates remain Ethanol is not added into the lysate before transferring sample mixture into FABG Mini Column Repeat the extraction procedure with a new sample Incorrect preparation of Wash Buffer 4 Ethanol is not added into Wash Buffer when first open Make sure that the correct volumes of ethanol 96 100 is added into Wash Buffer when first open Repeat the extraction procedure with a new sample 11 FABGK Trouble Shooting Problem Possible Reasons Solution A s0 A ratio of eluted DNA is low 5 The volume or the percentage of ethanol is not correct before adding into Wash Buffer Make sure that the correct volumes of ethanol 96 100 is added into Wash Buffer when first open Repeat the extraction procedure with a new sample 6 Genomic DNA is contaminated Do not wet the rim of the column during sample and buffer loading A s0 A ratio of eluted DNA is high 1 A lot of residual RNA in eluted DNA Follow the General Protocol step 2 to remove RNA 2 FABG Buffer added to the sample before adding RNase A Make sure that Rnase A has been added to the sample before adding FABG Buffer when using optional RNase step Degradation of eluted DNA 1 Sample is old Always use fresh or well stored sample for genomic DNA extraction
14. e pH of ddH O is between 7 5 9 0 Use Elution Buffer provided for elution Elution Buffer or ddH O is not completely absorbed by column membrane After Elution Buffer or ddH O is added stand the FABG Column for 5 minutes before centrifugation Brown residues B remain on the column membrane after washing Poor Cell Lysis It is because of insufficient Proteinase K activity Repeat the extraction procedure with a new sample Use a fresh or well stored Proteinase K stock solution Do not add Proteinase K stock directly to FABG Buffer It is because of insufficient mixing with FABG Buffer Repeat the extraction procedure with a new sample Mix the sample and FABG Buffer immediately and thoroughly by pulse vortexing Poor cell lysis because of insufficient incubation time Repeat the extraction procedure with a new sample Extend the incubation time and make sure that no residual particulates remain 9 FABGK Trouble Shooting Problem Possible Reasons Solution Brown residues remain on the column membrane after washing 2 Ethanol is not added into the lysate before transferring sample mixture into FABG Column Repeat the extraction procedure with a new sample 3 Incorrect preparation of Wash Buffer A Ethanol is not added into Wash Buffer when first open Make sure that the correct volumnes of ethanol 96 100 is added into Wash Buffer when
15. ep will avoid the residual liquid to inhibit subsequent enzymatic reactions Step 5 Elution 16 17 18 Place the dry FABG Column to a new 1 5ml microcentrifuge tube Add 100ul of Preheated Elution Buffer or TE to the membrane center of FABG Column Stand FAGB Column for 3 5 min or until the buffer is absorbed by the membrane Important Step For effective elution make sure that the elution solution is dispensed onto the membrane center and absorbed completely Centrifuge for 30 seconds at full speed 14 000 rpm or 10 000 x g to elute the DNA Standard volume for elution is 100 ul If sample has low number of cells reduce the elution volume 30 ul 50 ul to increase DNA concentration If higher DNA yield is required repeat the DNA Elution step to increase DNA recovery and the total volume could be 200 ul Step Final Pure DNA 19 Store the DNA fragment at 4 C or 20 C Special Protocol For Frozen Blood Step 1 Sample Preparation 1 Transfer up to 200uI blood to a 1 5ml microcentrifuge tube not provided If the sample volume is less than 200ul add the appropriate volume of PBS 2 Add 30ul Proteinase K 10 mg ml to the sample and briefly mix Then incubate for 15 minutes at 60 C Step 2 Cell Lysis 3 Add 200ul FABG Buffer to the sample and mix by vortex 4 Incubate in a 70 C water bath for 15 minutes to lyse the sample During incubation invert the sample every 3 minutes 5 Prehe
16. leated erythrocytes eg bird or fish can be up to 10ul ii Add 150 ul of FATG Buffer and the blood sample into a 1 5ml microcentrifuge tube not provided Mix by vortex and then follow Step 2 Cell Lysis Step 2 Cell Lysis 1 Add 200 ul of FABG Buffer to the sample and vortex for 5 seconds 2 Incubate for 10 minutes at 70 C or until the sample lysate is clear During incubation invert the tube every 3 minutes 3 Preheat required Elution Buffer for Step 5 DNA Elution in a 70 C water bath 4 Optional Step If RNA free genomic DNA is required add 5 l of 10 mg ml RNase A to the sample and mix by vortex Then incubate for 5 minutes at room temperature 5 Follow the General Protocol starting from Step 3 Binding Special Protocol For Bacteria Step 1 Sample Preparation A For Gram negative bacteria i Transfer the appropriate number of bacterial cell up to 1 x10 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 Add 200 ul of FATG Buffer and resuspend the pellet by vortex or pipetting Incubate for 5 minutes at room temperature iii Follow the Cultured Cell Protocol starting from Step 2 Cell Lysis B For Gram positive bacteria i Transfer the appropriate number of bacterial cell up to 1 x 10 to a 1 5ml microcentrifuge tube not provided and cenirifuge at full speed 14 000 rpm or 10 000 x g for 1
17. 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 or pipetting iii Incubate for 10 minutes at room temperature During incubation invert the tube every 2 3 minutes iv Follow the Cultured Cell Protocol starting from Step 2 Cell Lysis Special Protocol For Fungus Step 1 Sample Preparation i Harvest appropriate number of fungus cell up to 5 x 107 to a 1 5ml microcentrifuge tube not provided and centrifuge at 5 000 x g for 10 minute Then discard the supernatant ii Add 600 ul of sorbitol buffer 1 2 M sorbitol 10 mM CaCl 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 at 2 000 x g for 10 minutes to harvest the spheroplast and then remove the supernatant 9 v Add 200 ul of FATG Buffer to the tube and resuspend the cell pellet by vortex or pipetting vi Incubate at room temperature for 5 minutes and then follow the Cultured Cell Protocol starting from Step 2 Cell Lysis 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 bec
18. nd mix by vortex 8 Incubate for 10 minutes at room temperature or until the sample lysate is clear During incubation invert the tube every 3 minutes 9 Preheat required Elution Buffer for Step 5 DNA Elution in a 70 C water bath 10 Optional Step If RNA free genomic DNA is required add 5 pl of 10 mg ml RNase A to the sample and mix by vortex Then incubate for 5 minutes at room temperature Step 3 Binding 11 Add 200ul ethanol 96 100 to the sample and vortex for 10 seconds Pipetting if there is any precipitate 12 Place a FABG Column to a 2ml collection tube Transfer the sample mixture including any precipitate carefully to FABG Column Centrifuge for 5 minute at full speed 14 000 rpm or 10 000 x g and discard the 2ml collection tube Place the FABG Column in a new 2ml Collection tube Step 4 Washing 13 14 15 Wash FABG Column with 400ul W1 Buffer Centrifuge for 30 seconds at full speed 14 000 rpm or 10 000 x g and discard the flow through Place the FABG Column back in the 2ml Collection tube Wash FABG Column with 600ul Wash Buffer ethanol added Centrifuge for 30 seconds at full speed 14 000 rpm or 10 000 x g and discard the flow through Make sure that ethanol has been added into Wash Buffer when first open Place the FABG Column back in the 2ml Collection tube Centrifuge for an additional 3 min at full speed 14 000 rpm or 10 000 x g to dry the column Important Step This st
19. x thoroughly by pulse vortexing for 30 seconds 7 Briefly spin the tube to remove drops from the inside of the lid 8 Place a FABG Column to a collection tube Transfer the sample mixture including any precipitate carefully to FABG Column Centrifuge for 1 minute and discard the flow through then place FABG Column to a new Collection tube 5 FABGK 9 Immediately Wash FABG Column with 500 pl W1 Buffer by centrifugefor 1 minute then discard the flow through Make sure that ethanol has been added into W1 Buffer when first open 10 Wash FABG Column with 750 pl Wash Buffer by centrifuge for 1 minute then discard the flow through Make sure that ethanol has been added into Wash Buffer when first open 11 Centrifuge for an additional 3 min to dry the column Important Step This step will avoid the residual liquid to inhibit subsequent enzymatic reactions 12 Place FABG Column to Elution Tube 13 Add 100 200 plof Elution Buffer or ddH O pH 7 5 9 0 to the membrane center of FABG Column Stand FAGB Column for 3 min Important Step For effective elution make sure that the elution solution is dispensed onto the membrane center and is absorbed completely Standard volume for elution is 200 ul If sample has low number of cells reduce the elution volume 50ul 150ul to increase DNA concert ration 14 Centrifuge for 2 min to elute the DNA 15 Store the DNA fragment at 4 C or 20 C FABGK 6 Special
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