WizardÂ® Plus SV Minipreps DNA Purification System Technical ...

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III.ProtocolsMaterials to Be Supplied by the User(Solution compositions are provided in Section VI.)• LB agar plates containing antibiotic• LB medium containing antibiotic• ethanol (95%)• microcentrifuge capable of 14,000 × g• sterile 1.5ml microcentrifuge tubes• centrifuge capable of 10,000 × gPrior to using a new Wizard ® Plus SV Minipreps DNA Purification System,dilute the provided Wizard® Plus SV Column Wash Solution as follows:Add 7ml of 95% ethanol for a final volume of 11ml for the 10-prep system(Cat.# A1270).Add 35ml of 95% ethanol for a final volume of 55ml for the 50-prep system(Cat.# A1330 and A1340).Add 170ml of 95% ethanol for a final volume of 270ml for the 250-prep system(Cat.# A1460 and A1470).Note: An A 600 reading of2–4 ensures that cells havereached the proper growthdensity for harvesting andplasmid DNA isolation.A. Preparation of E. coli1. Use a single, well-isolated colony from a fresh Luria-Bertani (LB) agar plate(containing antibiotic) to inoculate 1–10ml of LB medium (containing the sameantibiotic). We recommend LB culture medium. Rich media, such as TerrificBroth, may produce very high cell densities that may overload the DNApurification system.2. Incubate overnight (12–16 hours) at 37°C in a shaking incubator. Incubationtime can be optimized to increase the plasmid DNA yield for a given culturevolume. However, it has been observed that as a culture ages the DNA yieldmay begin to decrease due to cell death and lysis within the culture.For high-copy-number plasmids, we do not recommend processing more than5ml of bacterial culture. If more than 5ml of culture is processed, the capacity of theWizard® Plus SV Minipreps Spin Column will be exceeded and no increase in plasmidyield will be obtained.For low-copy-number plasmids, it may be necessary to process larger volumesof bacterial culture (up to 10ml) for recovery of sufficient DNA. Processing greaterthan 10ml of culture will lead to insufficient clearing of the bacterial lysate andthus increased contaminants in the plasmid DNA.To prevent shearingof chromosomal DNA,Do Not vortex after Step2. Mix only by inverting thetubes.B. Production of a Cleared Lysate1. Harvest 1–5ml (high-copy-number plasmid) or 10ml (low-copy-number plasmid)of bacterial culture by centrifugation for 5 minutes at 10,000 x g in a tabletopcentrifuge. Pour off the supernatant and blot the inverted tube on a papertowel to remove excess media.2. Add 250µl of Cell Resuspension Solution and completely resuspend the cellpellet by vortexing or pipetting. It is essential to thoroughly resuspend thecells. If not already in a microcentrifuge tube, transfer the resuspended cells tosterile 1.5ml microcentrifuge tube(s).3. Add 250µl of Cell Lysis Solution and mix by inverting the tube 4 times (do notvortex). Incubate until the cell suspension clears, approximately 1–5 minutes.Note: It is important to observe partial clearing of the lysate before proceedingto addition of the Alkaline Protease Solution (Step 4); however, do notincubate longer than 5 minutes.Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Telephone 608-274-4330 · Fax 608-277-2516 · www.promega.comPart# TB225Page 4Printed in USA.Revised 3/01
4. Add 10µl of Alkaline Protease Solution and mix by inverting the tube 4 times.Incubate for 5 minutes at room temperature.Alkaline protease inactivates endonucleases and other proteins released duringthe lysis of the bacterial cells that can adversely affect the quality of the isolatedDNA.5. Add 350µl of Wizard® Plus SV Neutralization Solution and immediately mix byinverting the tube 4 times (do not vortex).6. Centrifuge the bacterial lysate at maximum speed (around14,000 x g) in amicrocentrifuge for 10 minutes at room temperature.C. Plasmid DNA Isolation and Purification ProtocolDo Notincubate for more than 5minutes with AlkalineProtease Solution at Step4, as nicking of the plasmidDNA may occur.The Wizard ® Plus SV Minipreps DNA Purification System allows a choice of methodsfor purification of plasmid DNA when systems with Miniprep Vacuum Adapters arepurchased (Cat.# A1340, A1470). Plasmid DNA may be purified from the bacteriallysate using microcentrifugation to force the cleared lysate through the Wizard®Plus SV Minipreps Spin Column and to wash the plasmid DNA. Alternatively, avacuum can be used to pull the lysate through the Spin Column and to wash theplasmid DNA. Miniprep Vacuum Adapters allow the use of a vacuum manifold (e.g., aVac-Man® Laboratory Manifold) and vacuum source for DNA purification.Centrifugation ProtocolPrepare plasmid DNA purification unitsby inserting one Spin Column into one2ml Collection Tube for each sample.1. Transfer the cleared lysate (approximately850µl, Section III.B, Step 6) tothe prepared Spin Column by decanting.Avoid disturbing or transferring anyof the white precipitate with the supernatant.2. Centrifuge the supernatant at maximumspeed in a microcentrifuge for 1 minuteat room temperature. Remove the SpinColumn from the tube and discard theflowthrough from the Collection Tube.Reinsert the Spin Column into theCollection Tube.3. Add 750µl of Column Wash Solution,previously diluted with 95% ethanol, tothe Spin Column.4. Centrifuge at maximum speed in amicro-centrifuge for 1 minute at roomtemperature. Remove the Spin Columnfrom the tube and discard theflowthrough. Reinsert the Spin Columninto the Collection Tube.Vacuum ProtocolAttach one Miniprep Vacuum Adapterwith Luer-Lok ® fitting to one port of themanifold (e.g., Promega’s Vac-Man® orVac-Man® Jr. Laboratory VacuumManifold). Insert a Spin Column intothe Miniprep Vacuum Adapter untilsnugly in place.1. Transfer the cleared lysate (approximately850µl, Section III.B, Step 6) tothe prepared Spin Column by decanting.Avoid disturbing or transferring anyof the white precipitate with the supernatant.2. Apply a vacuum of at least 15 inches ofmercury (Hg) to pull the liquid throughthe Spin Column. When all liquid hasbeen pulled through the column,release the vacuum.3. Add 750µl of the Column WashSolution, previously diluted with 95%ethanol, to the Spin Column.4. Apply a vacuum to pull the ColumnWash Solution through the SpinColumn. When all the liquid has beenpulled through the Spin Column,release the vacuum.Note: If the white precipitateis accidentally transferredto the Spin Column,pour the Spin Columncontents back into a sterile1.5ml microcentrifugetube and centrifuge foranother 5–10 minutes atmaximum speed. Transferthe resulting supernatantinto the same SpinColumn that was used initiallyfor this sample. TheSpin Column can bereused but only for thissample.Comparison of Inches ofHg to Other PressureMeasurements.15 Inches Hg50.8kPa381 Torr0.501atm7.37psi38.1cm Hg508mbarPromega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Telephone 608-274-4330 · Fax 608-277-2516 · www.promega.comPrinted in USA.Part# TB225Revised 3/01 Page 5
Page 3: II.Product ComponentsProduct Size C
Page 7 and 8: B. Choosing a Bacterial StrainEndon
Page 9 and 10: Table 2 outlines the amount of term
Page 11 and 12: VI.Composition of Buffers and Solut

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