FavorPrep Blood / Cultured Cell Genomic DNA
Contents
1. 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 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 up to 50 ug of total DNA depends on the sample types and the number of cells in the sample2. 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 For FABGK 001 1 100preps add 80 ml ethanol 96 100 to Wash Buffer when first open For FABG
3. K 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 Mix thoroughly by pulse vortexing for 30 seconds 7 Briefly spin the tube to remove drops from t
4. ain 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 2 Buffer for gel electrophoresis contaminated with DNase Use fresh running buffer for gel
5. eat 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 the pH of ddH O is between 7 5 9 0 Use Elution Buffer provided for elution Elution Buffer
6. 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
7. f 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 procedure with a new sample Extend the incubation time and make sure that no residual particulates rem
8. he 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 Protocol For cultured Cells 1 Harvest Cells a Cells grown in suspension i Transfer the appr
9. opriate 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 page 7 2 Poor cell lysis A it is because of insufficient Proteinase K activity Rep
10. 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 first open Repeat the extraction procedure with a new sample B The volumn or the percentage o
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