User Manual FavorPrep Genomic DNA Mini Kit (Blood/Cultured Cell)
Contents
1. clear During incubation invert the tube every 3 minutes 3 Briefly spin the tube to remove drops from the inside of the lid 4 Preheat Elution Buffer 200 ul per sample in a 70 C water bath for Step 4 DNA Elution 5 Optional Step If RNA free genomic DNA is required add 5yl of 10 mg ml RNase A to the sample and vortex then incubate for 5 minutes at room temperature 6 Follow the Fresh Blood Protocol starting from Step 3 Binding Cultured Cell Protocol Sample Preparation i 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 at 2 000 x g for 10 minutes to harvest the spheroplast v Remove the supernatant and add 200 ul of FATG Buffer to the tube and resuspend the cell pellet by vortex Incubate at room temperature for 5 minutes Follow the Cultured Cell Protocol starting from Step 1 Cell Lysis lt S vii2. 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 20 Incubate the FAGB Column at 37 C for 10 minutes in an incubator 21 Centrifuge for 1 minute to elute the DNA Standard volume for elution is 100 pl 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 22 Store the DNA fragment at 4 C or 20 C Cultured Cell Protocol Sample Preparation For Cultured Cells i Trypsinize the adherent cells before harvesting ii Transfer the appropriate number of cell up to 1 x 107 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 150 ul of RBC Lysis Buffer 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 10yl ii Add 150 pl of FATG Buffer and the blood sample into a 1 5ml microcentrifuge tube not provided then mix by vortex Step 1 Cell Lysis 1 Add 200 ul of FABG Buffer and mix by vortex for 5 seconds 2 Incubate for 10 minutes at 70 C or until the sample lysate is
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 preps RBC Lysis Buffer FATG Buffer FABG Buffer W1 Buffer Wash Buffer concentrated Elution Buffer FABG Column 2 ml Collection Tube FABGK 100 100 Preps 135 ml 30 mi 40 ml 45 ml 25 ml 30 ml 100 pcs 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 Fresh Blood Protocol e Step 1 RBC Lysis 1 Collect fresh human blood in an anticoagulant ireat collection tube 2 Transfer up to 300ul fresh blood to a microcentrifuge tube not provided If the blood sample is more than 300 ul 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 3000 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 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 Briefly spin the tube to remove drops from the inside of the lid 10 Preheat Elution Buffer 200 pl pe
4. col starting from Step 3 Binding Buffy Coat Protocol 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 1 RBC Lysis 1 Transfer up to 200ul buffy coat to a 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 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 binding Step 2 Cell Lysis 7 Add 250 ul of FABG Buffer 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 Briefly spin the tube to remove drops from the inside of the lid 10 Preheat Elution Buffer 200 ul per sampl
5. e in a 70 C water bath for Step 5 DNA Elution 11 Optional Step If RNA free genomic DNA is required add 5pl of 10 mg ml RNase A to the sample and vortex then incubate for 5 minutes at room temperature Step 3 Binding 12 Add 250ul ethanol 96 100 to the sample Mix thoroughly by pulse vortexing for 10 seconds Pipetting if there is any precipitate 13 Briefly spin the tube to remove drops from the inside of the lid 14 Place a FABG Column to a collection tube Transfer the sample mixture including any precipitate carefully to FABG Column Centrifuge for 5 minute and discard the flow through then place FABG Column to a new Collection tube Buffy Coat Protocol Step 4 Washing 15 Immediately Wash FABG Column with 400ul W1 Buffer ethanol added by centrifuge for 1 minute then discard the flow through Make sure that ethanol has been added into W1 Buffer when first open 16 Wash FABG Column with 600ul Wash Buffer ethanol added by centrifuge for 1 minute then discard the flow through Make sure that ethanol has been added into Wash Buffer when first open 17 Centrifuge for an additional 3 min to dry the column Important Step This step will avoid the residual liquid to inhibit subsequent enzymatic reactions Step 5 Elution 18 Place FABG Column to a new 1 5ml microcentrifuge tube 19 Add 100ul of Preheated Elution Buffer or TE to the membrane center of FABG Column Stand FAGB Column for 3 5 min or until
6. 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 20 Centrifuge for 30 seconds to elute the DNA Standard volume for elution is 100 pl If sample has low number of cells reduce the elution volume 30 ul 50 pl 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 21 Store the DNA fragment at 4 C or 20 C Frozen Blood Protocol Sample Preparation 1 Transfer up to 200ul sample to a 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 Mix thoroughly by pulse vortexing Incubate for 15 minutes at 60 C Step 1 Cell Lysis 3 Add 200ul FABG Buffer to the sample Mix thoroughly by pulse vortexing 4 Incubate at 70 C for 15 minutes to lyse the sample During incubation invert the sample every 3 5 minutes 5 Briefly spin the tube to remove drops from the inside of the lid 6 Preheat Elution Buffer 200 ul per sample in a 70 C water bath for Step 4 DNA Elution 7 Optional Step If RNA free genomic DNA is required add 5yl of 10 mg ml RNase A to the sample and vortex then incubate for 5 minutes at room temperature 8 Follow the Fresh Blood Proto
7. r sample in a 70 C water bath for Step 5 DNA Elution 11 Optional Step If RNA free genomic DNA is required add 5yl of 10 mg ml RNase A to the sample and vortex then incubate for 5 minutes at room temperature Step 3 Binding 12 Add 200ul ethanol 96 100 to the sample Mix thoroughly by pulse vortexing for 10 seconds Pipetting if there is any precipitate 13 Briefly spin the tube to remove drops from the inside of the lid 14 Place a FABG Column to a collection tube Transfer the sample mixture including any precipitate carefully to FABG Column Centrifuge for 5 minute and discard the flow through then place FABG Column to a new Collection tube Step 4 Washing 15 Immediately Wash FABG Column with 400ul W1 Buffer ethanol added by centrifuge for 30 seconds then discard the flow through Make sure that ethanol has been added into W1 Buffer when first open 16 Wash FABG Column with 600ul Wash Buffer ethanol added by centrifuge for 30 seconds then discard the flow through Make sure that ethanol has been added into Wash Buffer when first open 17 Centrifuge for an additional 3 min to dry the column Important Step This step will avoid the residual liquid to inhibit subsequent enzymatic reactions Fresh Blood Protocol Step 5 Elution 18 Place FABG Column to a new 1 5ml microcenirifuge tube 19 Add 100ul of Preheated Elution Buffer or TE to the membrane center of FABG Column Stand FAGB Column for 3 5
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