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

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C. Use of Alkaline ProteaseTo improve the quality of plasmid DNA isolated from both EndA+ and EndA–strains of E. coli, Promega’s Wizard ® Plus SV Minipreps DNA PurificationSystem includes an alkaline protease solution. Alkaline protease, originally identifiedas subtilisin Carlsberg, is isolated from the bacterium Bacillus licheniformis(1). Approximately 250µg are added per sample at the end of the lysis step,during the preparation of a cleared bacterial lysate to inactivate endonucleases.The alkaline protease also acts to nonspecifically degrade proteins, thus reducingthe overall level of protein contaminants in the cleared bacterial lysate (2,3).Alkaline protease is useful in this procedure because it is optimally active at apH of 9 and above, the conditions present during alkaline lysis procedure. Whenthe lysate is neutralized, alkaline protease activity is substantially reduced (4,5).The DNA prepared by this procedure has been tested extensively in a range ofmolecular biology applications including fluorescent sequencing, restrictionenzyme digestion and in cloning.D. Considerations for Automated Fluorescent SequencingNote: Optimal automatedfluorescent sequencingresults are routinelyobtained by using highcopy-numberplasmidsand EndA– strains ofE. coli.For the application of automated fluorescent sequencing, special considerationshould be given to the selection of plasmid type and E. coli strain to optimize yieldand plasmid quality.Purified plasmid DNA must be within the proper concentration range for successfulautomated cycle sequencing (ideally 0.2µg/µl and not less than 0.1µg/µl).When working with plasmid DNA from low-copy-number plasmids, we stronglyrecommend that DNA concentrations be determined by agarose gel/ethidiumbromide quantitation prior to any application (6). DNA quantitation by spectrophotometricmethods is prone to errors and requires a large amount of sample.The Wizard ® Plus SV Minipreps System routinely yields 3.5–5.0µg of plasmidDNA when using a pGEM® Vector (f) and DH5α cells in 1.5ml of LB medium.For low-copy-number plasmids, a larger culture volume is required to obtain sufficientDNA for sequencing. Typical low-copy-number plasmid yields are1.5–3.0µg of plasmid DNA from 10ml of LB culture medium, using thepALTER®-1 Vector(f,g) (ampicillin-resistant) and DH5α cells.Special Considerations for Sequencing Using BigDye ChemistryThe Wizard ® Plus SV Minipreps System yields template suitable for use in anumber of fluorescent dye sequencing methods, including ABI PRISM®BigDye terminator (The Perkin-Elmer Corporation Cat.# 4303149, 4303150,4303151) reactions.When performing dilutions of the BigDye terminator-ready reaction mix, it isessential to dilute the reaction mix using the appropriate dilution buffer (250mMTris-HCl [pH 9.0], 10mM MgCl 2 ).When using high-copy-plasmids, over 500 bases of readable sequence can beobtained from terminator-ready reaction mixes diluted as much as six-fold.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 8Printed in USA.Revised 3/01
Table 2 outlines the amount of terminator-ready reaction mix and dilution bufferrequired to obtain the appropriate dilution for BigDye terminator reactions. Fordetails on running these reactions, please refer to the protocol supplied with theBigDye terminator system. For each reaction, add the reagents in Table 2 to aseparate tube.Table 2. Appropriate Dilutions for BigDye Terminator Reactions.AmountComponent No Dilution 1:2 1:4 1:6terminator-readyreaction mix* 8.0µl 4.0µl 2.0µl 1.3µldouble-stranded plasmidDNA template 200–500ng 200–500ng 200–500ng 200–500ngprimer 3.2pmol 3.2pmol 3.2pmol 3.2pmoldilution buffer** 0µl 2.0µl 3.0µl 3.4µlNuclease-Free Waterto a final volume of 20µl 20µl 20µl 20µl* Terminatr-ready reaction mix is a 2.5X solution.** Dilution buffer is a 5X solution.V. TroubleshootingSymptoms Possible Causes CommentsPoor cell lysis Too many bacterial cells Use cultures grown to an A 600 ofin culture medium 2–4. All media should contain anantibiotic. Use only recommendedculture volumes for low-and highcopy-numberplasmids (10ml maximumfor low copy and 5ml for highcopy-numberplasmids).Poor resuspension of Thoroughly resuspend cell pelletsbacterial cell pellet prior to cell lysis. Vortex or pipetpellet with Cell ResuspensionSolution. No cell clumps should bevisible after resuspension.No plasmid DNA purified Ethanol was not added to Prepare the Column Wash SolutionColumn Wash Solution as instructed in Section IIIbefore beginning the procedure.Inaccurate quantitation of Quantitate plasmid DNA yield viaplasmid DNA yield agarose gel/ethidium bromideelectrophoresis.DNA floats out of well Be certain to allow the full tenduring loading of gel for minutes for drying (with vacuumquantitationprotocol) after the final wash stepto allow evaporation of anyremaining ethanol.Increase loading dye concentration.Low-copy-number Use high-copy-number plasmids.plasmid usedFor questions notaddressed here, pleasecontact your localPromega Branch Officeor distributor. Contactinformation available at:www.promega.com.E-mail:techserv@promega.comPromega 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 9
Page 3 and 4: II.Product ComponentsProduct Size C
Page 5 and 6: 4. Add 10µl of Alkaline Protease S
Page 7: B. Choosing a Bacterial StrainEndon
Page 11 and 12: VI.Composition of Buffers and Solut

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