User Manual FavorPrep Genomic DNA Mini Kit (Blood/Cultured Cell)
Contents
1. 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 5 Follow the General Protocol starting from Step 3 Binding Step 4 Binding 11 12 Add 250ul 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 400l 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 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 tube2. 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 Step 4 Washing 13 14 15 Wash FABG Column with 400l 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 step 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 Prehe
3. FavorPrep Genomic DNA Mini Kit Blood Cultured Cell User Manual Cat No FABGK 100 100 Preps FABGK 300 300 Preps For Research Use Only Kit Contents Cat No FABGK 100 FABGK 300 preps 100 Preps 300 Preps RBC Lysis Buffer 135 ml 405 ml FATG Buffer 30 ml 75 ml FABG Buffer 40 ml 100 ml W1 Buffer 45 ml 130 ml Wash Buffer concentrated 25 ml 50 ml Elution Buffer 30 ml 75 ml FABG Column 100 pcs 300 pcs 2 ml Collection Tube 200 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 buffy coat Up to 1 x 10 of Cultured animal cells Up to 1 x 10 of Cultured bacterial cells Up to 5 x 10 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 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
4. 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 Add 200 ul of FATG Buffer and resuspend the pellet by vortex or pipetting Incubate for 5 minutes at room temperature Follow the Cultured Cell Protocol starting from Step 2 Cell Lysis 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 centrifuge at full speed 14 000 rpm or 10 000 x g for 1 minute Then discard the supernatant 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 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 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 i Add 600 ul of sorbitol buffer 1 2 M sorbitol 10 mM CaCl 0 1 M Tris HCI pH 7 5 35MM B mercaptoethanol and resuspend the pellet iii Add 200 U of lyticase or zymolase Incubate for 30 minutes a
5. ated 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 Preheat 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 The
6. hanol 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 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 cenirifuge 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 nucleated 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
7. n 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 pl of RBC Lysis Buffer Mix the tube by vortex only Be sure the pellet is completely resuspended or the column would be barred 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 lysa
8. 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 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 e Sample is old Always use fresh or well stored sample for genomic DNA extraction e Residual ethanol contamination Afier Wash step centrifuge at 4 000 x g for an additional 10 minutes to dry the FABG Midi Column e RNA contamination Special Protocol For Bacteria Step 1 Sample Preparation A For Gram negative bacteria B Transfer the
9. t 30 C Centrifuge the mixture at 2 000 x g for 10 minutes to harvest the spheroplast and then remove the supernatant 9 Brief Procedure Cell lysis FASG 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 300ul 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 pl of FABG Buffer and 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 5pl 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 200uI et
10. te 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
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