NOTICE: Kits NP100010N and NP100011N do not
Contents
1. ORIGENE Please see notice below regarding kits NP100010N and NP100011N Overview and Intended Use The PowerPrep Express Plasmid Purification Systems are designed for rapid isolation of plasmid DNA A modified alkaline SDS procedure is used to create a cleared lysate containing plasmid DNA with minimal amounts of genomic DNA 1 The lysate is applied to a spin cartridge containing silica based membranes where the plasmid DNA is selectively adsorbed 2 Adsorption to the membranes is influenced by buffer composition and temperature RNA proteins and other cellular waste are removed with wash buffers containing alcohol Since the DNA is eluted at high concentration in TE Buffer no precipitation is necessary All solutions are passed through the column with brief centrifugations or vacuum allowing completion of the protocol in 30 to 45 min e Plasmid DNA up to 45 kb can be purified e Yields of up to 40 ug miniprep can be obtained Results are dependent on plasmid copy number plasmid type bacterial strain and growth conditions e g medium antibiotics temperature and aeration e The purified plasmid DNA is high quality and is suitable for most applications including automated fluorescent DNA sequencing manual DNA sequencing amplification reactions restriction mapping cloning and labeling References 1 Birnboim H and Doly J 1979 Nucleic Acids Res 7 1513 2 Vogelstein B and Gillespie D 1979 Proc2. nuclease rich bacteria are used e g endA strains 7 Cartridge Wash Place the spin cartridge back into the 2 mL wash tube Add 700 uL of Wash Buffer containing ethanol to the spin cartridge Centrifuge at 12 000 x g for 1 min Discard the flow through Centrifuge again at 12 000 x g for 1 min to remove residual wash buffer 8 Plasmid Elution Place the spin cartridge into a 1 5 mL re covery tube supplied Add 75 uL of warm TE Buffer directly to the center of the spin cartridge Incubate at room temperature for 1 min then centrifuge at 12 000 x g for 2 min NOTE Room temperature TE Buffer may be used for elution but optimal recovery is obtained with TE warmed to 65 to 70 C ii f EY f fy ef lt a Z r fry al Doll eX Z lt o Z 3 6 C lt _ 1MIN aa e P 9620 Me 888 267 a 301 340 Custsupport origene com www origene com Harvest Calls 250 ul Call Suspension Buffer 1 250 ul Call Lysis Sdiution 350 ul Neutralization Buffer A Load Cartridge 500 ul Optional Wash Buffer 4 700 ul Wash Buffer 75 ul TE Buffer wam 200 Rockville MD 20850 Page 4 of 5 Usage Notes Vacuum Protocol Perform all centrifugations at room temperature Before beginning a Preheat an aliquot of TE Buffer to 65 to 70 C b Verify that RNase A has been added to Cell Suspension Buffer and
3. that no precipitate has formed in Cell Lysis Solution c Verify that ethanol has been added to Wash Buffer and Optional Wash Buffer See Advance Preparations 1 Cell Harvesting Pellet 1 to 5 mL of an overnight culture Thoroughly remove all medium 2 Cell Suspension Add 250 uL of Cell Suspension Buffer containing RNase A to the pellet and suspend the cells until homogeneous 3 Cell Lysis Add 250 uL of Cell Lysis Solution Mix gently by inverting the capped tube five times Do not vortex Incubate at room temperature for exactly 5 min Longer incubation times may reduce yield 4 Neutralization Add 350 uL of Neutralization Buffer and mix immediately by inverting the tube until the solution is homogeneous When large cell pellets have been processed more vigorous shaking may be required Do not vortex Centrifuge the mixture at 12 000 x g for 10 min 5 Vacuum Manifold Preparation Attach the vacuum manifold to a vacuum source Attach a cartridge to a luer extension on the vacuum manifold Load the supernatant from step 3 into the cartridge Apply vacuum until all supernatant goes through the cartridge and then turn off the vacuum source 6 Optional Wash Add 500 uL of Optional Wash Buffer to the cartridge Apply vacuum until all supernatant goes through the cartridge and then turn off the vacuum source 7 Cartridge Wash Add 700 uL of Wash Buffer containing ethanol to the cartridge Apply vacuum until all superna
4. Natl Acad Sci USA 76 615 Safety and Use Statement This product contains hazardous reagents It is the end user s responsibility to consult the applicable MSDS s before using this product Disposal of waste organics acids bases and radioactive materials must comply with all appropriate federal state and local regulations If you have any questions concerning the hazards associated with this product please call OriGene at 888 267 4436 All biological materials should be handled as potentially hazardous Follow universal precautions as established by the Centers for Disease Control and Prevention and by the Occupational Safety and Health Administration when handling and disposing of potentially infectious or hazardous agents This product is authorized for laboratory research use only The product has not been qualified or found safe and effective for any human or animal diagnostic application Uses other than the labeled intended use may be a violation of applicable law 9620 Medical Center Drive Suite 200 Rockville MD 20850 888 267 4436 phone 301 340 9254 fax Custsupport origene com www origene com Page 1 of 5 PowerPrep Express Plasmid Purification Kits Store at RT Catalog No NP100010 11453 016 50 Reactions Catalog No NP100011 11453 024 250 Reactions Components included with this kit MINIPREP kits Components NP100010 NP100011 Reactions 50 250 Cell Susp
5. e that RNase A has been added to Cell Suspension Buffer The bacteria should be resuspended completely by vortexing or pipetting up and down until no cell clumps remain 2 Lysis Mix gently but thoroughly Do not vortex this will shear the genomic DNA The lysate should appear viscous Do not allow the lysis reaction to proceed for more than 5 min 3 Neutralization After addition of Neutralization Buffer a fluffy white material should form and the lysate becomes less viscous The precipitated material contains genomic DNA proteins cell debris and SDS The lysate should be mixed thoroughly to ensure even precipitation If the mixture still appears to contain a gelatinous and slightly brownish material more mixing is required to completely neutralize the solution This is more likely to happen when large cell pellets have been processed Mit i Harvest Calls 4 250 ul Cell Suspension Buffer 4 SMIN 250 ul Cell Lysis 7 Solution 4 350 ul Neutralization Buffer 4 Load Cartridge Ga y an 4 ci i 500 ul Optional Wash Buffer 4 700 pl Wash Buffer 75 ul TE Buffer warm 9620 Medical Center Drive Suite 200 Rockville MD 20850 888 267 4436 phone 301 340 9254 fax Custsupport origene com www origene com Page 3 of 5 Usage Notes Centrifugation Protocol Perform all centrifugations at room temperature Before beginning a Preheat an aliquot
6. ension Buffer 15 mL 65 mL RNase A 100 uL 650 pL Cell Lysis Solution 15 mL 65 mL Neutralization Buffer 20 mL 90 mL Wash Buffer 12 mL 55 mL Optional Wash Buffer 12 mL 55 mL TE Buffer 15 mL 30 mL Spin Cartridges or Columns 50 each 250 each e Recovery Tubes 50 each 250 each Additional Materials Required e 95 100 ethanol e 65 to 70 C water bath e For minipreps microcentrifuge capable of reaching gt 12 000 x g NOTICE Kits NP100010N and NP100011N do not include any buffers listed A protocol detailing how to create buffers necessary is provided Storage Conditions Store all components at Room Temperature After the addition of RNase A to the Cell Suspension Buffer store buffer at 4 C Advance Preparations 1 Add RNase A to Cell Suspension Buffer according to the following instructions Volume of RNase A to Volume of Cell add to Cell Suspension Suspension Buffer p Buffer 15 mL 85 uL 65 mL 375 uL 120 mL 650 uL These instructions are also on the label of the bottle Mix well Place a mark on the label to indicate that RNase A has been added Store Cell Suspension Buffer with RNAse A at 4 C for up to six months 2 Add ethanol to Wash Buffer according to the following instructions Wash Buffer Volume of Wash Volume of EtOH to add to Buffer Wash Buffer 12 mL 30 mL 55 mL 140 mL 95 mL 235 mL These instructions are also on the label of the bottle Mix well P
7. lace a mark on the label to indicate that ethanol has been added Add ethanol to Optional Wash Buffer according to the following instructions Optional Wash Buffer Volume of Optional Wash Buffer 12 mL 55 mL Volume of EtOH to add to Optional Wash Buffer 17 mL 75 mL These instructions are also on the label of the bottle Mix well Place a mark on the label to indicate that ethanol has been added 3 Check Cell Lysis Solution for precipitate If necessary warm the solution briefly at 37 C to dissolve the precipitate 9620 Medical Center Drive Suite 200 Rockville MD 20850 888 267 4436 phone 301 340 9254 fax Custsupport origene com www origene com Page 2 of 5 Critical Parameters e Use volumes temperatures incubation times and centrifugations precisely as indicated in the protocols Store columns and solutions at recommended temperatures e Grow cultures in LB medium Cell density should be 1 to 1 5 Asoo units mL or 1 x 10 cells per mL e Do not overload the columns Use the recom mended culture volumes as indicated in the protocol to obtain optimal purity e For centrifugation of maxiprep columns use a swinging bucket rotor to ensure complete removal of solutions Important Considerations for the Alkaline Lysis and Neutralization Steps 1 Cell Suspension For efficient lysis it is important to use a vessel that is large enough to allow complete mixing of the lysis buffers Ensur
8. of TE Buffer to 65 to 70 C b Verify that RNase A has been added to Cell Suspension Buffer and that no precipitate has formed in Cell Lysis Solution c Verify that ethanol has been added to Wash Buffer and Optional Wash Buffer See Advance Preparations 1 Cell Harvesting Pellet 1 to 5 mL of an overnight culture Thoroughly remove all medium 2 Cell Suspension Add 250 uL of Cell Suspension Buffer containing RNase A to the pellet and suspend the cells until homogeneous 3 Cell Lysis Add 250 uL of Cell Lysis Solution Mix gently by inverting the capped tube five times Do not vortex Incubate at room temperature for exactly 5 min Longer incubation times may reduce yield 4 Neutralization Add 350 uL of Neutralization Buffer and mix immediately by inverting the tube until the solution is homogeneous When large cell pellets have been processed more vigorous shaking may be required Do not vortex Centrifuge the mixture at 12 000 x g for 10 min 5 Cartridge Loading Place a spin cartridge in a 2 mL wash tube Load the supernatant from step 4 into the spin cartridge Centrifuge at 12 000 x g for 1 min Discard the flow through 6 Optional Wash Place the spin cartridge back into the 2 mL wash tube Add 500 uL of Optional Wash Buffer to the spin cartridge Incubate at room temperature for 1 min Centrifuge at 12 000 x g for 1 min Discard the flow through NOTE This wash is recommended with the miniprep pro tocol when
9. one 301 340 9254 fax Custsupport origene com www origene com Page 5 of 5 Suggested Solution Carefully remove all medium before suspending cells in step 2 Ensure the full volume of Neutralization Buffer is added when neutralizing the lysate in step 4 Do not process more cells than recommended over an individual column Process additional cells over additional columns Ensure that TE Buffer is warmed to 65 to 70 C Reduce the elution volume This will give higher DNA concentrations but will also give a decrease in yield Precipitate plasmid DNA and dissolve in a smaller volume of TE Buffer Invert tubes to mix when adding Cell Lysis and Neutralization Buffers Mix thoroughly but gently to homogeneity Do not vortex Incubate the lysate at room temperature for no longer than 5 min at step 3 Permanently denatured DNA will appear as a band electrophoresing just ahead of the supercoiled plasmid DNA This material will not be digested by restriction endonucleases Use correct volume of Cell Suspension Buffer Ensure that RNase A was added to Cell Suspension Buffer and that Cell Suspension Buffer containing RNase A is stored at 4 C and is less than 6 months old For minipreps discard wash buffer flow through prior to the second centrifugation in step 7
10. tant goes through the cartridge and then turn off the vacuum source Place the cartridge into a 2 mL wash tube Centrifuge at 12 000 x g for 1 min to remove residual wash buffer 8 Plasmid Elution Place the cartridge into a 1 5 mL re covery tube supplied Add 75 uL of warm TE Buffer directly to the center of the cartridge Incubate at room temperature for 1 min then centrifuge at 12 000 x g for 2 min NOTE Room temperature TE Buffer may be used for elution but optimal recovery is obtained with TE warmed to 65 to 70 C Appendix A Troubleshooting Guide Problem Possible Cause Low yield of DNA Lysate at improper pH or salt concentration for binding to column Low copy number plasmid Incomplete DNA elution Plasmid DNA concentration too low Genomic DNA sheared in handling Chromosomal DNA contamination Additional plasmid forms Plasmid DNA permanently present denatured RNA contamination RNase A digestion incomplete Inhibition of enzymatic Ethanol in the DNA eluate reaction Technical Support For further technical assistance please contact us at 888 267 4436 or by email at techsupport origene com Technical support and troubleshooting guides for these products can also be found on our website at www origene com Related Products To see our full line of PowerPrep purification products visit our website at www origene com 9620 Medical Center Drive Suite 200 Rockville MD 20850 888 267 4436 ph
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