User Manual FavorFilter Endotoxin-Free Plasmid DNA
Contents
1. PM3 Buffer at 4 C before use Additional Requirements 1 50 ml centrifuge tube 2 Isopropanol 3 70 ethanol Brief Procedure i Culture bacteria cells Harvest bacteria cells Resuspend PM1 Buffer Centrif cnnaae C Cell Lysis PM2 Buffer Neutralize PM3 Buffer Add PTR Buffer Incubate on ice for 30 min Equilibrate Plasmid i Maxi Columns by gravity flow PEQ Buffer Filtration N DNA Binding Washing PW Buffer DNA Elution PEL Buffer by gravity flow Precipitate DNA Washing Dissolve DNA Centrifuge C Cr Ce N General Protocol 1 Harvest the bacterial culture up to 240 ml by centrifugation at 6 000 x g for 15 minutes Note For culture volume more than 240 ml add twice the amount of PM1 Buffer RNase A added PM2 Buffer and PM3 Buffer for the following steps 2 Place a PM Maxi Column onto a 50 ml cenirifuge tube 3 Equilibtate a PM Maxi column by applying 12 5 ml of PEQ Buffer Allow the column to empty by gravity flow 4 Discard the filtrate 5 Apply 20 ml of PM1 Buffer RNase A added to resuspend the cell pellet by vortexing or pipetting 6 Add 20 ml of PM2 Buffer and mix genily by inverting the tube 15 times Do not vortex and avoid shearing genomic DNA 7 Incubate for 5 minutes at room temperature until lysate clears 8 During the incubation prepare the FavorFilter Cartridge e Remove the cap from the tip of the cartridge and pull o2. Winterchim FavorFilter Endotoxin Free Plasmid DNA Extraction Maxi Kit User Manual Cat No FAFTE 001 EF 4 preps For Research Use Only v 1005 1 Introduction The FavorFilter Endotoxin Free Plasmid DNA Extraction Maxi Kit is designed for rapid and efficient extraction of high quality plasmid DNA With provided filter cartridges the bacteria lysates will be removed without centrifugation Following a gravity flow procedure the plasmid DNA is bound to the resin and the contaminants can be remove with wash buffer After using this convenient kit the purified plasmid DNA is suitable for downstream application such as transfection in vitro transcription and translation and all enzymatic modification Specification Sample Size 60 240 ml of bacteria for high copy number plasmid 200 480 ml of bacteria for low copy number plasmid Binding Capacity up to 1 5 mg of DNA Kit Contents AT 4 preps PEQ Buffer 55 ml PM1 Buffer 85 ml PM2 Buffer 85 ml PM3 Buffer 85 ml PTR Buffer 25 mi PW Buffer 130 ml PEL Buffer 65 ml RNase A 50mg ml 170 ul Favorfilter Maxi Cartridge 4pcs PM Maxi Column 4 pcs Brief spin the RNase A tube and adding the RNase A to PM1 Buffer Store the PM1 Buffer at 4 C after adding RNase A 1 Important Notes 1 Store the PM1 Buffer RNase A added at 4 C after use 2 If precipitates have formed in PM2 Buffer warm the buffer in 37 C waterbath to dissolve preciptates 3 Pre chill
3. th 5 ml of room temperature 70 ethanol Then shake the tube gently Centrifuge at 20 000 x g for 10 minutes at 4 C e Centrifuge speed should not be less than 20 000 x g Carefully remove the supernatant Then air dry the DNA pellet until the tube is completely dry Or incubate the DNA pellet at 70 C for 10 min Dissolve the DNA pellet in 300 ul or a suitable volume of TE or ddH20 Troubleshooting Low yield Bacterial cells were not lysed completely Too many bacterial cells were used e After PM3 Buffer addition break up the precipitate by inverting to ensure higher yield Purified DNA dose not perform well in downstream application RNA contamination Prior to using PM1 Buffer ensure that RNase A was added If RNase A added PM1 Buffer is overdue add additional RNase A Too many bacterial cells were used reduce the sample volume Genomic DNA contamination e Do not use overgrown bacterial culture e During PM2 and PM3 Buffer addition mix gently to prevent genomic DNA shearing www interchim com Ba interchim 211 bis Avenue Kennedy BP 1140 Agence Paris Normandie 03103 Montlu on France 33 0 1 41 32 34 40 33 0 4 70 03 88 55 Fax 33 0 1 47 91 23 90 Fax 33 0 4 70 03 82 60 e mail interchim paris interchim com e mail interchim interchim com
4. ut the plunger e Place the cap back to the tip of the cartridge and stand the cartridge vertically in a suitable rack 9 Add 20 ml of PM3 Buffer and mix immediately by inverting the tube 10 times Do not vortex Proceed directly to step 10 10 Pour the lysate into the barrel of the FavorFilter Cartridge Add 5 ml of PTR Buffer and mix gently by pipetting incubate the lysate on ice for 30 minutes After the incubation the sample mixture will become clear Important Step To ensures filtration without clogging 10 minutes incubation is essential to make the precipitate float up 11 12 13 14 15 16 17 18 19 20 21 Remove the cap from the tip of the FavorFilter Cartridge Gently insert the plunger into the FavorFilter Cartridge and filter the lysate into the equilibrated PM Maxi column then allow it to flow through by gravity flow Discard the filtrate Wash the PM Maxi column by applying 30 ml of PW Buffer Allow the column to empty by gravity folw Discard the filtrate Place PM Maxi column onto a clean 50 ml centrifuge tube not provided and add 15 ml of PEL Buffer to elute DNA by gravity flow Precipitate DNA by adding 11 ml of isopropanol to the eluted DNA from Step 15 Mix well by inverting the tube 10 times Centrifuge at 20 000 x g for 30 minutes at 4 C e Centrifuge speed should not be less than 20 000 x g Carefully remove the supernatant and wash the DNA pellet wi
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