User Manual FavorFilter Plasmid DNA Extraction
Contents
1. C after adding RNase A 2 If precipitates have formed in PM2 Buffer warm the buffer in 37 C waterbath to dissolve preciptates 3 Pre chill PM3 Buffer at 4 C before use Additional Requirements 1 50 ml centrifuge tube 2 Isopropanol 3 70 ethanol Brief Procedure Harvest bacteria cells Resuspend Cell Lysis centrifuge N Culture bacteria cells A _ Neutralize Ka Filtration gt DNA Binding i Washing B DNA Elution Precipitate DNA i E Washing centrifuge E Dissolve DNA 2 General Protocol 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 centrifuge 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 gently by inverting the tube 15 times Do not vortex and avoid shearing genomic DNA 7 Incubate for 5 minutes at room temperature uniil lysate clears 8 During the incubation prepare the FavorFilter Cartridge e Remove the cap from the tip of the cartridge and pull out the plunger e Place the cap back to the t2. Q s P FAVORGEN FavorFilter Plasmid DNA Extraction Maxi Kit User Manual Cat No FAFTE 001 4 preps FAFTE 001 1 10 preps For Research Use Only v 1005 1 Australian distributors Fisher Biotec Australia free call 1800 066 077 EE etree email info fisherbiotec com ae web www fisherbiotec com Introduction The FavorFilter 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 centri fugation 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 e FAFTEOO1 FAFTEOO1 1 Kit Contents 4 ha 10 at PEQ Buffer 55 ml 135 ml PM1 Buffer 85 ml 215 ml PM2 Buffer 85 ml 215 ml PM3 Buffer 85 ml 215 ml PW Buffer 130 ml 165 mi x 2 PEL Buffer 65 ml 215 ml RNase A 50mg ml 170 ul 430 ul FavorFilter Maxi Cartridge 4pcs 10 pcs PM Maxi Column 4pcs 10 pcs Important Notes 1 Brief spin the RNase A tube and adding the RNase A to PM1 Buffer Store the PM1 Buffer at 4
3. ip 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 Incubate the lysate for 10 minutes at room temperature 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 with 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 sh
4. ould 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
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