User Manual FavorPrep Plasmid DNA Extraction Midi
Contents
1. volume Genomic DNA contamination 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 ethanol2. Winterchim FavorPrep Plasmid DNA Extraction Midi Kit User Manual Cat No FAPDE 002 25 Preps FAPDE 002 1 50 Preps For Research Use Only v 1005 1 Introduction FavorPrep Plasmid DNA Extraction Midi Kit is designed for efficient extraction of high quality plasmid DNA from 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 up to 60 ml of bacteria for high copy number plasmid up to 120 ml of bacteria for low copy number plasmid Binding Capacity 650 ug column Kit Contents FAPDE 002 FAPDE 002 1 25 preps 50 preps PEQ Buffer 135 ml 135 ml x 2 PM1 Buffer 215 ml 215 mi x2 PM2 Buffer 215 ml 215mlx 2 PM3 Buffer 215 ml 215mlx 2 PW Buffer 165 mi x 2 165 mi x 4 PEL Buffer 215 ml 215mlx 2 RNase A 50 mg ml 430 ul 430 wl x 2 PM Midi Column 25 pcs 50 pcs Important Notes 1 Brief spin the RNase A tube and adding the RNase A to PM1 Buffer Store 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 di
3. ssolve preciptates Additional Requirements 1 50 ml centrifuge tube 2 Isopropanol 3 70 ethanol Brief Procedure Culture bacteria cells A x C Harvest bacteria cells Resuspend PM1 centrifuge Lyse PM2 J Neutralize PM3 Equilibrate Plasmid F Midi Columnis by rJ gravity flow v DNA Bindin nan g PEQ Buffer Washing PW Buffer 2 DNA Elution PEL Buffer Precipitate DNA if Washing centrifuge Dissolve DNA s Pure plasmid DNA W General Protocol Place a PM Midi Column into a 50 ml centrifuge tube Add 5 ml of PEG Buffer to equilibrate the PM Midi column and allow the column to empty by gravity flow Discard the filtrate Harvest the bacterial culture by centrifugation at 6 000 x g for 15 minutes Add 8 ml of PM1 Buffer RNase A added to resuspend the cell pellet by vortexing or pipetting Add 8 ml of PM2 Buffer and mix gently by inverting the tube 15 times Do not vortex to avoid shearing genomic DNA Incubate for 3 minutes at room temperature until lysate clears Add 8 ml of PM3 Buffer and mix immediately by inverting the tube 10 times Do not vortex Centrifuge at 15 000 x g for 20 minutes at 4 C e Centrifuge speed should not be less than 15 000 x g Transfer the supernatant to the equilibrated PM Midi Column and allow the column to empty by gravity flow Discard the filtrate 9 Add 12 ml of PW Buffer to wash the PM Midi column and allow the col
4. umn to empty by gravity flow Discard the filtrate 10 Place the PM Midi column into a clean 50 ml centrifuge tube not provided and add 8 ml of PEL Buffer to elute DNA by gravity flow 11 Precipitate DNA by adding 6 ml of isopropanol to the eluted DNA from previous step Mix well by inverting the tube 10 times 12 Centrifuge at 20 000 x g for 30 minutes at 4 C e Centrifuge speed should not be less than 20 000 x g 13 Carefully remove the supernatant and wash the DNA pellet with 5 ml of room temperature 70 ethanol 14 Centrifuge at 20 000 x g for 10 minutes at 4 C e Centrifuge speed should not be less than 20 000 x g 15 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 16 Dissolve the DNA pellet in 300 ul or a suitable volume of TE buffer 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 DNA failed o precipitate or DNA pellet was lost after precipitation 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
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