User Manual FavorPrep Endotoxin-Free Plasmid DNA Extraction
Contents
1. FavorPrep Endotoxin Free Plasmid DNA Extraction Maxi Kit User Manual Cat No FAPDE 003 EF 10 Preps For Research Use Only v 1005 1 Introduction The FavorPrep Endotoxin Free Plasmid DNA Extraction Maxi Kit is designed for efficient extraction of high quality plasmid DNA from 50 250 ml of bacterial culture This kit provide the alkaline lysis reagents and the columns packed with anion exchanger resin After the cells lysis the plasmid DNA is bound to the resin insided the column by a gravity flow procedure 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 FAPDE0O03 EF 10 preps PEQ Buffer 135 ml PM1 Buffer 215 ml PM2 Buffer 215 ml PM3 Buffer 215 ml PTR Buffer 55 ml PW Buffer 165 ml x 2 PEL Buffer 215 ml RNase A 50 mg ml 430 ul PM Maxi Column 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 C after adding RNase A 2 If precipitates have formed in PM2 Buffer warm the buffer in 37 C waterbath to dissolve preciptates Additional Requirements 1 50 ml cent2. es at 4 C e Centrifuge speed should not be less than 15 000 x g 8 Transfer the supernatant from step 7 to a clean 50 ml centrifuge Add 5 ml of PTR Buffer and mix gently by inverting the tube 10 times 9 Incubate on ice for 30 minutes e After the incubation the sample mixture will become clear 10 18 Transfer the the sample mixture from step 9 to the equilibrated PM Maxi Column and allow the column to empty by gravity flow Discard the filtrate Add 30 ml of PW Buffer to wash the PM Maxi column and allow the column to empty by gravity flow Discard the filtrate Place the PM Maxi column into 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 previous step 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 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 pl or a suitable volume of TE or ddH20 Troubleshooting Low
3. rifuge tube 2 Isopropanol 3 70 ethanol Brief Procedure Culture bacteria cells N Harvest bacteria cells N me i Resuspend PM1 centrifuge 4 Lyse PM2 Neutralize PM3 Add endotoxin remove Buffer PTR Buffer gt on ice for 30 minutes Equilibrate Plasmid a Midi Columnis by DNA Binding A Washing PW Buffer gravity flow DNA Elution PEL Buffi PEQ Buffer lution uffar Precipitate DNA Washing Dissolve DNA centrifuge Pure plasmid DNA nN aj CT CMH General Protocol Please Read Important Note Before Starting The Following Steps 1 Place a PM Maxi Column into a 50 ml centrifuge tube Add 12 5 ml of PEQ Buffer to equilibrate the PM Maxi column and allow the column to empty by gravity flow Discard the filtrate 2 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 3 Add 20 ml of PM1 Buffer RNase A added to resuspend the cell pellet by vortexing or pipetting 4 Add 20 ml of PM2 Buffer and mix gently by inverting the tube 15 times Do not vortex to avoid shearing genomic DNA 5 Incubate for 5 minutes at room temperature until lysate clears 6 Add 20 ml of PM3 Buffer and mix immediately by inverting the tube 10 times Do not vortex 7 Centrifuge at 15 000 x g for 20 minut
4. yield Bacterial cells were not lysed completely Too many bacterial cells were used e After PM3 Buffer addition break up the precipitate by inverting DNA failed to precipitate or DNA pellet was lost after precipitation e DNA pellet was insufficiiently redissolved Purified DNA dose not perform well in downstream application RNA contamination e Make sure that that RNase A was has been added in PM1 Buffer when first using 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 e Lysis time was too long over 5 minutes Too much salt residual in DNA pellet Wash the DNA pellet twice with 70 ethanol
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