40min Fast Plasmid Maxiprep Kit - Syringe Format
Contents
1. Order amp Inquiry Tel 713 732 2181 Fax 1 866 747 4781 E mail order biotool com vs biotool com Save time Save funding Note e Buffer B1 precipitates below 10 C warm up at 50 C to dissolve if precipitates e Shake gently and don t incubate over 10 min to avoid genomic DNA fragmentation which greatly hampers plasmid DNA purity 4 Add 3 mL Buffer N3 mix completely by inverting the tube 10 times then shaking vigorously for 5 times It is critical to mix the solution well More shaking is required to completely neutralize the solution until the mixture appears congealed brownish or viscous 5 Set the Filter Syringe upon a new 50 mL conical tube and pour the lysate into the barrel of the filter syringe Let stand at room tempera ture for 1 to 2 min until the white precipitates float to the top Insert the plunger and expel the clear lysate DNA remains in the clear lysate into the tube Stop when the syringe encounters strong resistance Flip the syringe upside down and the precipitate will float to the top Expel the volume again to get maximal clear lysate Small amounts of lysate may remain in the precipitate and can be ignored Optional Low speed centrifuge After adding buffer N3 spin down the lysate mixture at 3500 rpm for 1 to 2 min in a 50 mL conical tube to remove most of the precipitate The DNA will remain in the super natant which is transferred to the filter syringe The plunger can2. then be pushed more easily to filter the lysate 6 Add 0 7 1 0 volume Buffer RET e g 14 to 20 mL of Buffer RET to 20 mL of clear lysate and 10 mL 100 ethanol into the lysate Mix well by vigorous shaking for 5 times The solution mixture MUST be immediately filtered through the BtBind DNA Columns Note Immediate filtration is critical to avoid salt precipitation 7 Use a vacuum pump to suck the lysate ethanol mixture through the column to absorb the plasmid DNA onto the membrane Optional Set the assembled DNA column in an upright position upon a 50 mL conical tube using the Plastic Wrench as the column holder Add the lysate ethanol mixture into a DNA column and use the plunger to expel the mixture through the column Take the plunger out of the DNA column and add the remaining mixture Expel the plunger until all of the mixture goes through the DNA binding membrane Note There is no need for disassembling the membrane column before taking the plunger out of the DNA column Order amp Inquiry Tel 49 89 46148500 Fax 49 89 461485022 E mail eu order biotool com 8 Add 10 mL DNA Wash Buffer into DNA column Hold the plunger and invert the syringe upside down several times to wash the cavity Expel wash buffer using the plunger or a vacuum pump 9 Repeat Step 8 10 Use the plastic wrench to detach the membrane from the DNA column and insert the membrane into a 2 0 mL Microfuge tube Spin the column
3. als listed below e 100 ethanol e High speed centrifuge Equipped for 1 5 mL or 2 mL EP tubes Order amp Inquiry Tel 49 89 46148500 Fax 49 89 461485022 E mail eu order biotool com Lysis and neutralization Clear lysate through a syringe filter U U q figure 1 Flow chart of protocol Bind DNA to the DNA column Wash the column with DNA Wash Buffer p Detach the column and insert to a 2 0 mL tube Spin the column to dry and elute the DNA 1 Harvest 150 250 mL fresh bacterial overnight culture at 37 C for 14 16 h by vigorous shaking with initial inoculation ratio of 1 500 to 1 1000 by centrifugation at 5 000 g for 10 min at room temperature Pour off the supernatant and blot the inverted tube on paper towels to completely remove residual medium Note e Complete removal of residual medium is critical for bacteria lysis in the next steps Do not use more than 250 mL culture If processing gt 250 mL of bacterial culture the buffers MUST be scaled up 2 Add 10 mL Buffer A1 and completely resuspend bacterial pellet by vortexing or pipetting Ensure that RNase A has been added into Buffer A1 before use Note Complete resuspension is critical for optimal yield 3 Add 9 mL Buffer B1 mix thoroughly by inverting 10 times with gentle shaking Incubate for 5 to 10 min at room temperature to obtain a clear lysate The mixture of completely lysed bacteria will appear transparent
4. at 12 000 rpm for 1 min discard the flow and spin again at 12 000 rpm for 2 min Air dry air blast in fume hood prefered the membrane at room temperature for 3 5 min 11 Insert the membrane into a new sterile 1 5 mL or 2 0 mL Micro fuge tube Add 500 uL Endofree Elution Buffer or ddH2O Pre warmed at 60 C to the center of the membrane and incubate for 1 min at room temperature Centrifuge at 12 000 rpm for 1 min to elute the DNA Add the DNA eluate back onto the membrane for a second elution Note The first elution normally yields about 60 70 of the DNA and the second elution yields another 30 of the DNA Optional To obtain the maximum recovery add another 300 uL Endofree Elution Buffer or ddH2O to the center of the membanre and incubate for 1 min at room temperature Elute the DNA by centrifugation at 12 000 rom for 1 min The final DNA concentration will decrease with the increased volume Order amp Inquiry Tel 713 732 2181 Fax 1 866 747 4781 E mail order biotool com Trouble shooting RA bio tool com Save time Save funding Possible Reason Suggested Improvement No DNA Plasmid lost in Host E coli Prepare fresh culture Low Yield Low Yield Low Yield Genomic DNA contamination ODze60 280 gt 2 0 Low ratio of OD260 230 Plasmid DNA floats out of wells while running in agarose gel DNA doesn t freeze or smell of ethanol Bacterial culture overgrown or not
5. fresh Low copy number plasmid Poor Cell lysis Improper manipulation after adding Buffer B1 RNA contamination Presence of contaminants EDTA phenol salt etc which absorbs at 230 nm Ethanol traces were not completely removed from column Order amp Inquiry Tel 49 89 46148500 Grow bacterial 12 16 hours Spin down culture and store at 20 C until use Scale up culture volume and the volume of Buffers A1 B1 N3 and 100 ethanol proportionally e Resuspend pellet thoroughly by vortexing and pipetting prior adding Buffer B1 e Make fresh Buffer B1 if the cap had not been closed tightly e Don t vortex or mix aggressively e Don t incubate over 10 minutes Add RNase A into Buffer A1 and Store at 4 C e Pre warm Buffer RET at 50 C for 15 min to avoid salt precipitation e Increase washing times e Air dry the membrane longer or in the ventilation system Make sure that no ethanol remains in the silicon membrane before eluting the plasmid DNA Re centrifuge or vacuum again if necessary Fax 49 89 461485022 E mail eu order biotool com
6. g biotool com Save time Save funding 40min Fast Plasmid Maxiprep Kit Syringe Format Description The Biotool endofree system uses a specially formulated buffer that extracts the endotoxin from bacterial lysate The final endotoxin level is lt 0 3 EU ug plasmid DNA Purified endofree DNA is ready for high performance downstream applications such as transfection of robust cells endotoxin sensitive cell lines primary cultured cells or embryo microinjection Components Cat B22213 Cat B22216 BtBind DNA Columns 25 100 DNA Wash Buffer 2x54 mL 10x54 mL Endofree Elution Buffer Notes for handling buffers Buffer A1 amp RNase A Spin down and add RNase A into Buffer A1 mix well and store at 4 C Buffer B1 Buffer B1 precipitates below 10 C Warm up Buffer B1 at 50 C if precipitates before use Keep the cap of Buffer B1 tightly closed after use Buffer N3 Buffer N3 may precipitate below 10 C Warm up Buffer N3 at 37 C if precipitates before use DNA Wash Buffer Add 216 mL 100 ethanol according to bottle label before use and mix well Endofree Elution Buffer Pre warm the Elution Buffer at 60 C to increase plamsmid DNA yield Order amp Inquiry y Tel 713 732 2181 Fax 1 866 747 4781 E mail order biotool com Storage e After added RNase A Buffer A1 should be stored at 4 C e All other materials can be stored at room temperature 20 26 C e The guaranteed shelf life is 12
7. months from the date of purchase Notice Below is Steps where mistakes are easily made Please read the following carefully Step 5 When transferred into Filter Syringe let stand the lysate at room temperature for 1 to 2 min until the white precipitates float to the top then press the plunger e Optional Spin down the lysate at 3500 rpm for 1 to 2 min to remove most of the precipitates and then transfer the supernatant into the Filter Syringe Step 6 Buffer RET is recommended to be pre warmed at 50 C to dissolve salt precipitates if any exists e The solution mixture MUST be immediately filtered to avoid salt precipitation after adding Buffer RET and 100 ethanol Step 7 e Use a vacuum pump to suck the solution mixture through the column to absorb the plasmid DNA onto the membrane Optional Transfer the solution mixture into the BtBind DNA column and press the plunger to expel the solution to bind DNA onto the membrane Step 11 e Endofree Elution Buffer or ddH2O must be pre warmed at 60 C to effectively elute the plasmid DNA Protocol The kit is optimized for high copy number plasmid purification Scale up both the volume of bacterial culture and buffers for low copy number plasmids The yield of plasmid DNA depends on the origin of the replication and the size of the plasmid Ensure all the buffers are handled as described above critical for efficient plasmid purification and provide materi
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