PhoenIX™ Midiprep Kit
Contents
1. protocol Centrifuge tubes that can hold gt 24 ml of liquid and can withstand 12 000 x g for low copy protocol lsopropanol 70 ethanol RNaseA and T1 optional supplement for high volume preps 3 Safety Precautions several components contain compounds that may cause irritation or burning when in contact with human tissue or during inhalation Wear personal protective equipment to prevent skin contact e g gloves lab coat and eye protection and prevent inhalation of reagent vapors and consumption of liquid during use Consult the enclosed Material Safety Data Sheet for additional details 4 Things to Know before Using the PhoenIX Midiprep Kit 4 1 Read Protocol Thoroughly before Use For optimal yield and purity of plasmid DNA it is critical to closely follow the protocol as directed Although the process of bacterial cell growth and lysis may be able to withstand many short cuts each time a step is not performed optimally the risk of an unacceptable result increases 4 2 High Copy vs Low Copy Plasmids High copy plasmids 3 5 ug DNA ml LB Medium should be grown in a culture volume of 15 50 ml If working with low copy plasmids 0 2 1 ug DNA ml LB Medium the larger culture volume 51 100 ml requires the use of twice as much of the alkaline lysis buffers The PhoenIX Midiprep Kit contains enough buffer for 25 high copy preps or 12 low copy preps If you plan to work exclusively with low copy plasmid2. for 5 minutes at 4 C 20 Completely remove ALL of the supernatant from the pellet with a pipette 21 Air dry the pellet for 10 minutes Note Drying with a vacuum chamber is not recommended because over dried DNA may be difficult to completely resuspend 22 Dissolve the plasmid DNA in 200 ul of water or TE Buffer DNA is immediately ready for downstream applications and may be moved to a smaller tube if desired 6 Detailed Protocol for Low Copy Plasmids 51 mi 100 ml Bacterial Culture Before beginning verify that RNase A red cap has been added to the Cell Resuspension Buffer yellow cap label and that no precipitate can be seen in the Lysis Buffer blue cap label See Sections 4 3 and 4 4 1 Place PhoeniX Midi column in the assembled Disposable Column Rack Place a waste container underneath that will hold gt 50 ml 2 Add 10 mi of Equilibration Buffer gray cap label to the surface of the column and allow the liquid to drain by gravity flow Note It will take 10 15 minutes for the column to drain completely During this process proceed to Step 3 10 visit us on the web at www qbiogene com anIX Midiprep Kit 3 Pellet 51 100 ml of a 1 5 x 10 cells ml bacterial culture see section 4 3 by centrifugation at 6 000 x g for 15 minutes at room temperature 4 Remove all traces of liquid medium from the bacterial cell pellet with a pipette Note f traces of culture medium remain with the cells the rati
3. meet our specification By acceptance of the product Buyer indemnifies and holds Qbiogene Inc harmless against and assumes all liability for the consequence of its use or misuse by the Buyer its employees or others including but not limited to the cost of handling Said refund or replacement is conditioned on Buyer notifying Qbiogene Inc within thirty 30 days of receipt of product Failure of Buyer to give said notice within thirty 30 days shall constitute a waiver by the Buyer of all claims hereunder with respect to said material s 11 Trademarks and Patents BIO 1018 Systems and RPM are registered trademarks of Qbiogene MiniPrep Express Molecular Biology Certified PhoenIX and RapidPURE are trademarks of Qbiogene PhoenIX M plasmid purification materials are covered under U S Patent 5 843 312 and foreign equivalents 16 visit us on the web at www qbiogene com Appendix I PhoenIX Midiprep Quick Reference Protocol Protocol Step Low Copy Midiprep Add Equilibration Buffer to Column Resuspend Pellet in Cell Resuspension Buffer with RNase A Add Lysis Buffer 8 mi Add Neutralization Buffer 8 mi Apply Lysate to Column 24 ml Add Column Wash Buffer Add Elution Buffer Wash with 70 EtOH Resuspend DNA Appendix Il PhoenIX Midiprep Diagnosis Protocol Starting Notes If a high copy plasmid that is expected to yield a high amount of plasmid DNA still does not produce enough DNA after the sugg
4. support the PhoenIX Midi Columns through the use of the cardboard Disposable Midi Column Rack Refer to Figure 1 First fold the rack along the lines as shown Starting with Tab A insert each tab into its corresponding slot as shown Refer to Figure 2 for a photograph of the assembled rack Figure 1 Assembly of Column Rack Figure 2 Assembled Column Rack 5 Detailed Protocol for High Copy Plasmids 15 mi 50 ml Bacterial Culture Before beginning verify that RNase A red cap has been added to the Cell Resuspension Buffer yellow cap label and that no precipitate can be seen in the Lysis Buffer blue cap label See Sections 4 3 and 4 4 1 Place PhoenIX Midi column in the assembled Disposable Column Rack Place a waste container underneath that will hold gt 50 ml 2 Add 10 mi of Equilibration Buffer gray cap label to the surface of the column and allow the liquid to drain by gravity flow Note It will take 10 15 minutes for the column to drain completely During this process proceed to Step 3 3 Pellet 15 50 ml of a 1 5 x 10 cells ml bacterial culture see section 4 3 by centrifugation at 6 000 x g for 15 minutes at room temperature 4 Remove all traces of liquid medium from the bacterial cell pellet with a pipette 8 visit us on the web at www qbiogene com Note f traces of culture medium remain with the cells the ratio of salts and pH values will not be optimal in subsequent steps 5 Add 4 ml o
5. 1 4 and run on a 0 8 agarose gel Expected results for Diagnosis Samples 1 5 significant amounts of DNA can normally be expected only in Diagnosis Samples 1 original cleared lysate and 5 eluate Diagnosis Sample 1 from the cleared lysate represents the amount of DNA present in 1 ml of the bacte rial culture It allows an estimation of the overall plasmid content and yield of the culture If yield is lower than expected optimization of the culture conditions medium temperature time etc may be required Diagnosis Sample 2 from the lysate flow through represents the amount of DNA that did not bind to the PhoenIX Midi Column Only degraded RNA should be visible No or only a faint DNA band should be seen on the gel If you get a DNA band nearly as intense as in Diagnosis Sample 1 the cleared lysate was likely not at the right salt concentration or pH in order to allow optimal DNA binding Follow the guidelines in the Troubleshooting section of the instruction manual If necessary the volume of Neutralization Buffer can be reduced by 10 Diagnosis Sample 3 from the first column wash should only contain degraded RNA and some or no traces of plasmid DNA Diagnosis Sample 4 from the second column wash should not contain more than traces of nucleic acid If you encounter significant amounts of DNA in the wash fractions the salt content of the Column Wash Buffer may be too high Ensure the correct bottle was used or pr
6. DNA with residual RNA might occur even in the presence of RNase Increasing the volume of Neutralization Buffer in step 8 of the protocol by 10 can minimize contaminating RNA without significantly decreasing plasmid DNA yield 8 Recommended Reference Format for Publications Plasmid DNA was purified using the PhoenIX Midiprep Kit Qbiogene Inc CA 9 Related Products 9 1 Plasmid Purification Kits Purification Scale Cat Kit Name Size Gigaprep 2075 400 PhoenIX Gigaprep Kit 5 preps Maxiprep 2075 300 PhoeniXTM Maxiprep Kit 25 preps 2075 350 PhoenIlX M Maxiprep Low Copy Refill Kit 12 preps Midiprep 2075 200 Phoen X M Midiprep Kit 25 preps 2075 250 PhoelX Midiprep Low Copy Refill Kit 12 preps Miniprep 2067 200 RapidPURE M Plasmid Mini Kit 60 preps 2067 400 RapidPURE Plasmid Mini Kit 120 preps 2067 600 RapidPURE Plasmid Mini Kit 300 preps 2067 200 RapidPURE M Plasmid Mini 96 Kit 96 preps 2067 400 RapidPURE Plasmid Mini 96 Kit 4 x 96 preps 2069 400 Yeast RPM Kit 100 preps 2070 200 RPM Kit 60 preps 2070 400 RPM Kit 120 preps 2070 500 RPM Kit 300 preps 2070 600 RPM Kit 600 preps 2000 200 MiniPrep Express Matrix 1 250 preps 2002 200 96well Prep Express 4 x 96 preps visit us on the web at www qbiogene com 15 PhoeniX Midipre 9 2 Molecular Biology Certified Growth Media BIO 101 Systems is well known for providing an excellent selection of high quality growth media in a variety of formula
7. PhoeniX Midiprep Kit lon exchange purification of 45 100 ug plasmid DNA from 15 100 ml of E coli cells Revision 2075 200 4E01 enlX Midiprep Kit PhoeniX Midiprep Kit lon exchange purification of 45 100 ug plasmid DNA from 15 100 mi of E coli cells Application Manual Revision 2075 200 4E01 Catalog 2075 200 25 high copy preps 12 low copy preps Storage Unopened box should be stored at ambient temperature 15 30 C Once RNase A has been added to Cell Resuspension Buffer store at 4 C and use within 6 months visit us on the web at www gbiogene com 3 2 2 2 4 1 4 2 4 3 4 4 4 0 4 6 4 7 11 TABLE OF CONTENTS Introduction Kit Components and User Supplied Materials Phoen X M Midiprep Kit Components User Supplied Materials Safety Precautions Things to Know before Using the PhoenIX Midiprep Kit Read Protocol Thoroughly before Use High Copy vs Low Copy Plasmids Growth of Bacterial Cells Prepare Cell Resuspension Buffer by adding RNase A Precipitate Material in Lysis Buffer Loading of PhoenIX Midi Columns Assembly of Disposable Midi Column Rack Detailed Protocol for High Copy Plasmids 15 ml 50 ml Bacterial Culture Detailed Protocol for Low Copy Plasmids 51 ml 100 ml Bacterial Culture Troubleshooting Low yields of plasmid DNA Slow column flow Chromosomal DNA contamination Multiple plasmid forms seen on agarose gel RNA contamination Recommended R
8. ash Buffer 850 mM NaCl 100 mM sodium acetate pH 4 0 is prepared gravimetrically using 8 203 g Sodium acetate 15 g of Glacial acetic acid 99 and 49 67 g of Sodium chloride After adjusting the water high quality to a final volume of 1 0 L the final pH value should be 4 0 0 1 The higher salt concentration and the lower pH value in the modified wash buffer allow better removal of residual RNA without significantly affecting the DNA yield Note f any drops of liquid from the cleared lysate added in step 13 remain on the plastic walls of the column wash them down into the column with the 10 ml of Column Wash Buffer 14 Repeat the wash in step 13 with a second application of 10 ml of Column Wash Buffer 5 8 minutes orange cap label 15 Discard the flow through Replace the waste collection container with a new centrifuge tube that can hold gt 12 ml of liquid and can withstand 12 000 x g 16 Add 5 ml of Elution Buffer pink cap label to the top of the column Allow the eluate to drain by gravity flow 5 minutes into the centrifuge tube Note If working with a large construct cosmid BAC YAC or P1 DNA elution of DNA can often be enhanced by pre warming the Elution Buffer to 50 C before use 17 Add 3 5 ml of Isopropanol to the eluted plasmid DNA in the centrifuge tube Mix and centrifuge at 12 000 x g for 30 minutes at 4 C Note Especially large DNA species cosmids BACs YACs or P1 DNA are very sticky and can spread o
9. eference Format for Publications Related Products Plasmid Purification Kits Molecular Biology Certified Growth Media Product Use Limitation Warranty Trademarks and Patents Appendix I PhoeniX Midiprep Quick Reference Protocol Appendix Il PhoenIX Midiprep Diagnosis Protocol Appendix Ill PhoenIXT Midiprep Buffer Compositions 4 visit us on the web at www gbiogene com niX Midiprep Kit 1 Introduction PhoenIX plasmid purification systems from Qbiogene rely on ion exchange chromatography through a unique patented solid phase column Precisely balanced pH values and salt concentrations permit optimal purification of highly clean plasmid DNA from the unique ion exchange resin Unlike resins found in other ion exchange kits PhoenIX columns are distinct First the smaller pore size of the resin particles increases the surface area available for binding Second the spacer molecule linking the positively charged amino group to the resin particles is longer than in other resins allowing a better degree of separation between different biomolecules during the binding and washing process The PhoenIX Midiprep Kit and PhoeniX Maxiprep Kit rely on centrifugation to clear the bacterial lysate and on gravity to pull liquids through the column material The PhoenIX Gigaprep Kit is driven by vacuum and offers lysate clearing via a filtration cartridge Plasmid DNA purified with PhoenIX columns can be used
10. epare new wash buffer Contact Qbiogene s Technical Support Department Diagnosis Sample 5 from the eluate represents the final amount of plasmid DNA recovered The plasmid DNA in the eluate should be free of genomic DNA RNA and be mostly supercoiled If you get no DNA in the eluate and little to no DNA in the lanes 2 3 and 4 ensure the correct bottle was used or prepare new elu tion solution Contact Qbiogene s Technical Support Department 20 visit us on the web at www qbiogene com hoeniX Midiprep Kit Appendix Ill PhoeniIX Buffer Compositions Cell Resuspension Buffer 50 mM Tris HCI pH 8 0 10 mM EDTA Lysis Buffer 200 mM NaOH 1 0 SDS w v Neutralization Buffer 3 1 M potassium acetate pH 5 5 Equilibration Buffer 600 mM NaCl 100 mM sodium acetate pH 5 0 0 15 Triton X 100 Column Wash Buffer 800 mM NaCl 100 mM sodium acetate pH 5 0 Elution Buffer 1250 mM NaCl 100 mM Tris pH 8 5 visit us on the web at www qbiogene com 21 PhoeniX Midiprep Kit NOTES 2 visit us on the web at www qbiogene com
11. estions in section 7 1 are followed perform the PhoenIX Midiprep Diagnosis Protocol to determine if the problem lies with the supplied reagents or the procedure itself Before beginning verify that RNase A red cap has been added to the Cell Resuspension Buffer yellow cap label and that no precipitate can be seen in the Lysis Buffer blue cap label See Sections 4 3 and 4 4 Diagnosis Protocol 1 Place PhoenIX Midi column in the assembled Disposable Column Rack Place a waste container underneath that will hold gt 50 ml visit us on the web at www qbiogene com 17 PhoenIX Midiprep 2 Add 10 mi of Equilibration Buffer gray cap label to the surface of the column and allow the liquid to drain by gravity flow Note It will take 10 15 minutes for the column to drain completely During this process proceed to Step 3 3 Pellet 25 ml of a 1 5 x 10 cells ml bacterial culture see section 4 3 by centrifugation at 6 000 x g for 15 minutes at room temperature 4 Remove all traces of liquid medium from the bacterial cell pellet with a pipette Note f traces of culture medium remain with the cells the ratio of salts and pH values will not be optimal in subsequent steps 5 Add 4 ml of RNase A containing Cell Resuspension Buffer yellow cap label to the cell pellet and vortex until completely resuspended 6 Transfer resuspended cells to a centrifuge tube that can hold gt 12 ml of liquid and can withstand 12 000
12. f RNase A containing Cell Resuspension Buffer yellow cap label to the cell pellet and vortex until completely resuspended 6 Transfer resuspended cells to a centrifuge tube that can hold gt 12 ml of liquid and can withstand 12 000 x g 7 Add 4 ml of Lysis Buffer blue cap label and securely cap the tube Mix thoroughly by inverting until the lysate appears to be homogeneous 5 6 inversions DO NOT VORTEX 8 Incubate 5 minutes at room temperature Note Do not incubate for longer than 5 minutes or plasmid DNA might become irreversibly denatured 9 Add 4 ml of Neutralization Buffer green cap label Securely cap the tube and mix immediately by multiple inversions until a homogeneous suspension containing no viscous matter is obtained DO NOT VORTEX Note f preparing several samples at once thoroughly mix each sample immediately after the addition of the Neutralization Buffer before adding the buffer to the next tube 10 Centrifuge the thoroughly mixed sample at 12 000 x g for 10 minutes at room temperature Note f a room temperature centrifuge is not available and sample is centrifuged at 4 C the super natant must be allowed to warm to room temperature 18 25 C prior to loading on the PhoenIX Midi Column 11 Verify that the Equilibration Buffer has been collected in the waste container beneath the Phoen X Midi Column Discard the flow through and replace the container 12 Use a pipette to remove the cleared ly
13. ged sample and add to the top of the equilibrated column Allow the lysate to drain by gravity flow 10 12 minutes Note Do not pour lysate directly onto the column Use a pipette to ensure that precipitate particles do not enter the column and cause clogging 14 Mix the flow through of the column well and remove 480 ul to a separate 1 5 ml microcentrifuge tube THIS IS DIAGNOSIS SAMPLE 2 Discard the remainder of the flow through and replace the collection container 15 Add 10 ml of Column Wash Buffer orange cap label to the top of the column and allow the liquid to drain by gravity flow Mix the flow through of the column well and remove 900 ul to a separate 1 5 ml microcentrifuge tube THIS IS DIAGNOSIS SAMPLE 3 Discard the remaining flow through and replace the empty collection container Note If any drops of liquid from the cleared lysate added in step 13 remain on the plastic walls of the column wash them down into the column with the 10 ml of Column Wash Buffer 16 Repeat the wash in step 15 with a second application of 10 ml of Column Wash Buffer orange cap label Mix the flow through of the column well and remove 900 ul to a separate 1 5 ml microcentrifuge tube THIS IS DIAGNOSIS SAMPLE 4 Note Do not add the second application of Column Wash Buffer until the first wash has run completely through the column 17 Discard the remaining flow through from the second wash and replace the collection container with a new ce
14. immediately in a wide variety of downstream applications including automated fluorescent sequencing enzymatic amplification labeling transcription cloning and other enzymatic manipulations Additionally the levels of endotoxin that remain after purifica tion are so low that successful transfection will occur with even sensitive cell lines Two protocols are included in this instruction manual The high copy protocol allows isolation of plasmid DNA from bacterial cultures of 15 ml to 50 ml The low copy protocol allows isolation of plasmid DNA from bac terial cultures of 51 ml to 100 ml In either protocol plasmid DNA is ready to precipitate in just over an hour 2 Kit Components and User Supplied Materials 2 1 PhoenlIX Midiprep Kit Components Description Quantity RNase A red cap 11 mg Cell Resuspension Buffer yellow cap label 110 ml Lysis Buffer blue cap label 110 ml Neutralization Buffer green cap label 110 ml PhoenIX Midi Columns 25 each Disposable Midi Column Rack 1 each Equilibration Buffer gray cap label 260 ml Column Wash Buffer orange cap label 525 ml Elution Buffer pink cap label 135 ml User Manual 1 each MSDS 1 each Certificate of Analysis 1 each visit us on the web at www qbiogene com 5 PhoenIX Midipre 2 2 User Supplied Materials Liquid collection container that can hold gt 50 ml Centrifuge tubes that can hold gt 12 ml of liquid and can withstand 12 000 x g for high copy
15. iscous matter is obtained DO NOT VORTEX Note f preparing several samples at once thoroughly mix each sample immediately after the addition of the Neutralization Buffer before adding the buffer to the next tube 10 Centrifuge the thoroughly mixed sample at 12 000 x g for 10 minutes at room temperature Note f a room temperature centrifuge is not available and sample is centrifuged at 4 C the super natant must be allowed to warm to room temperature 18 25 C prior to loading on the PhoenIX Midi Column 11 Verify that the Equilibration Buffer has been collected in the waste container beneath the PhoenIXTM Midi Column Discard the flow through and replace the container visit us on the web at www qbiogene com 11 PhoenlX Midipre 12 Use a pipette to remove the cleared lysate supernatant from the centrifuged sample and add to the top of the equilibrated column Allow the lysate to drain by gravity flow 10 12 minutes Discard the flow through and replace the empty container Note Do not pour lysate directly onto the column Use a pipette to ensure that precipitate particles do not enter the column and cause clogging 13 Add 10 ml of Column Wash Buffer orange cap label to the top of the column and allow the liquid to drain by gravity flow 5 8 minutes Note If working with a large construct cosmid BAC YAC or P1 DNA from a high volume culture removal of residual RNA may be more efficient if a modified Column W
16. ntrifuge tube that can hold gt 15 ml of liquid and can withstand 12 000 x g 18 Add 5 ml of Elution Buffer pink cap label to the top of the column Allow the eluate to drain by gravity flow 5 minutes into the centrifuge tube 19 Mix the eluted solution well and remove 900 ul to a separate 1 5 ml microcentrifuge tube THIS IS DIAG NOSIS SAMPLE 5 20 If completion of the protocol is desired add 3 5 ml of Isopropanol to the remainder of the eluted solu tion in the centrifuge tube Mix and centrifuge at 12 000 x g for 30 minutes at 4 C Continue with steps 17 21 of the main protocol in section 5 visit us on the web at www qbiogene com 19 PhoeniX Midipref Precipitation of Diagnosis Samples Diagnosis samples 1 5 must be precipitated with 0 7 volumes of isopropanol After centrifugation in a chilled microcentrifuge for 30 min at 14 000 x g allow all liquid to drain from the pellets by inverting the open tubes for at least 5 min on a sheet of absorbent paper towel Washing the pellets with 70 ethanol is not necessary Dry the nucleic acids further under vacuum for 5 10 minutes Do not over dry the pel lets Dissolve the nucleic acids in 30 50 ul water or TE buffer for 10 minutes at 37 C Testing of Diagnosis Samples 1 5 Remove 50 of the volume from Diagnosis Sample 5 from the elutate and save for spectrophotometric analysis Add electrophoresis loading dye to the remainder of Sample 5 and to all of samples
17. o of salts and pH values will not be optimal in subsequent steps 5 Add 8 mi of RNase A containing Cell Resuspension Buffer yellow cap label to the cell pellet and vortex until completely resuspended Note Normally the concentration of RNase A in the Cell Resuspension Buffer 100 ug ml should be suf ficient to remove all traces of RNA If you are working with a large construct cosmid BAC YAC or P1 DNA in 100 ml of bacterial culture it may be necessary to increase the amount of RNase A in the Cell Resuspension Buffer yellow cap label to 400 ug ml with user supplied RNase A The Cell Resuspension Buffer can be additionally supplemented with 100 U ml of RNase T1 The com bined activities of RNase A and T1 will result in a better digestion efficiency of the bacterial RNA thus leading to a better removal of the RNA during the column procedure 6 Transfer resuspended cells to a centrifuge tube that can hold gt 24 ml of liquid and can withstand 12 000 x g 7 Add 8 ml of Lysis Buffer blue cap label and securely cap the tube Mix thoroughly by inverting until the lysate appears to be homogeneous 5 6 inversions DO NOT VORTEX 8 Incubate 5 minutes at room temperature Note Do not incubate for longer than 5 minutes or plasmid DNA might become irreversibly denatured 9 Add 8 ml of Neutralization Buffer green cap label Securely cap the tube and mix immediately by multiple inversions until a homogeneous suspension containing no v
18. s the PhoenIX Midiprep Low Copy Refill Kit Cat 2075 250 is also available from Obiogene Large con structs cosmids BACs YACs and P1 DNA behave like very low copy plasmids and additional protocol mod fications are noted in the Low Copy Protocol Section 6 4 3 Growth of Bacterial Cells Inoculate 5 ml of selective medium with a single colony from a freshly streaked selective agar plate Incubate at least 8 hours at 37 C with vigorous shaking 300 rpm Dilute the starter culture 1 500 to 1 1000 For example add 100 ul of a high copy plasmid starter culture to 50 ml of fresh selective media Grow E coli cells at 37 C overnight with vigorous shaking to a density between 1 5 x 10 cells per ml of medium 1 1 5 ODgog units ml visit us on the web at www qbiogene com enIX Midiprep Kit 4 4 Prepare Cell Resuspension Buffer by adding RNase A To resuspend lyophilized RNase A red cap add 1 0 ml of Cell Resuspension Buffer yellow cap label and vortex Remove the entire volume of resuspended RNase A and add it to the bottle of Cell Resuspension Buffer Mix well and check the Contains RNase A box on the label Prepared Cell Resuspension Buffer Should be stored at 4 C and used within 6 months Qbiogene strongly recommends preparing the entire volume of Cell Resuspension Buffer at once However if it is certain that all of the prepared Cell Resuspension Buffer will not be used within 6 months a partial ba
19. sate supernatant from the centrifuged sample and add to the top of the equilibrated column Allow the lysate to drain by gravity flow 10 12 minutes Discard the flow through and replace the empty container Note Do not pour lysate directly onto the column Use a pipette to ensure that precipitate particles do not enter the column and cause clogging 13 Add 10 ml of Column Wash Buffer orange cap label to the top of the column and allow the liquid to drain by gravity flow 5 8 minutes Note If any drops of liquid from the cleared lysate added in step 13 remain on the plastic walls of the column wash them down into the column with the 10 ml of Column Wash Buffer visit us on the web at www qbiogene com 9 PhoeniX Midipre 14 Repeat the wash in step 13 with a second application of 10 ml of Column Wash Buffer 5 8 minutes orange cap label 15 Discard the flow through Replace the waste collection container with a new centrifuge tube that can hold gt 12 ml of liquid and can withstand 12 000 x g 16 Add 5 ml of Elution Buffer pink cap label to the top of the column Allow the eluate to drain by gravity flow 5 minutes into the centrifuge tube 17 Add 3 5 ml of Isopropanol to the eluted plasmid DNA in the centrifuge tube Mix and centrifuge at 12 000 x g for 30 minutes at 4 C 18 Pour out the supernatant taking care not to disturb the DNA pellet 19 Add 3 ml of 70 ethanol and wash the pellet Centrifuge at 12 000 x g
20. tch can be prepared instead Resuspend the RNase A as described above and add the appropriate amount to a smaller volume of Cell Resuspension Buffer For example instead of adding 1 ml of resus pended RNase A to 109 ml of buffer add 0 5 ml of resuspended RNase A to 54 5 ml of buffer The pre pared Cell Resuspension Buffer should be stored at 4 C as directed above The remaining volume of resus pended RNase A solution can be stored at 20 C for up to 2 years 45 Precipitate Material in Lysis Buffer If the PhoenIX M Midiprep Kit was shipped or stored at a low temperature a harmless precipitate may form in the Lysis Buffer blue cap label If a precipitate is seen incubate the bottle in a 37 C water bath for sev eral minutes and mix to bring the precipitate back into solution 4 6 Loading of PhoenIX Midi Columns Do not overload the PhoeniXTM Midi Columns with excess bacterial culture Up to 100 ml of bacterial cul ture can be used as long as the low copy protocol is followed If purification of other quantities of plasmid is desired PhoenIX M plasmid purification kits are available in Maxiprep and Gigaprep scales See the Related Products section for more information visit us on the web at www qbiogene com 1 PhoeniX Midiprep N 4 7 Assembly of Disposable Midi Column Rack The purchase of a separate column rack is not necessary with the PhoenIX M Midiprep Kit Qbiogene pro vides a convenient and inexpensive way to
21. tions We specialize in formulations for yeast and bacterial genetics and offer more than a thousand recipes and variations Each product is subjected to extensive quantitative testing and is Molecular Biology Certified through qualitative molecular biology based tests such as cell density and plasmid yield Choose from a wide variety of convenient packaging formats Pre mixed powders are avail able in capsule form single use pouches or in bulk sizes Liquid media pre poured plates and custom packaging options are also available If you prefer to make your own media BIO 101 Systems can supply you with raw materials such as tryptones and peptones yeast extract a wide variety of sugars and salts antibiotics and other media additives 10 Product Use Limitation amp Warranty Unless otherwise indicated this product is for research use only Purchase of Qbiogene Inc products does not grant rights to reproduce modify or repackage the products or any derivative thereof to third parties Qbiogene Inc makes no warranty of any kind expressed or implied including merchantability or fitness for any particular purpose except that the products sold will meet our specifications at the time of delivery Buyer s exclusive remedy and the sole liability of Qbiogene Inc hereunder shall be limited to at our dis cretion no replacement or compensation product credits refund of the purchase price of or the replace ment of materials that do not
22. uffer will have a significant negative impact on these critical parameters Ensure all culture media is removed with a pipette prior to resuspending cells in step 5 of the protocol 7 1 3 Alkaline lysis reagents were not added precisely Plasmid DNA will not bind optimally to the PhoenIX Midi Column unless it is at the correct salt concen tration and pH Measure volumes of alkaline lysis reagents precisely to ensure best results 7 1 4 DNA pellet was over dried Once a DNA pellet is over dried it can be difficult if not impossible to completely resuspend Air dry the pellet as directed in the protocol and do not use a vacuum pump system 7 1 5 Variability in plasmid copy number The total quantity of plasmid DNA in a particular E coli host cell is influenced by many variables The range of plasmid DNA per ml culture can vary from 0 2 ug ml low copy to gt 5 0 ug ml high copy The size and sequence of specific DNA inserts will often influence the copy number of a particular plasmid in some cases the copy number of a normally high copy plasmid will be reduced with a different insert visit us on the web at www qbiogene com 13 PhoeniX Midipre Qbiogene offers two protocols in this instruction manual one for high copy plasmids and one for low copy plasmids If a high copy plasmid gives a DNA yield that is lower than expected it may help to use a larger culture volume and follow the low cop
23. ut over the wall of the centrifuge tube if a fixed angle rotor is used If possible use a swinging bucket rotor or siliconized centrifuge tubes 18 Pour out the supernatant taking care not to disturb the DNA pellet 19 Add 3 ml of 70 ethanol and wash the pellet Centrifuge at 12 000 x g for 5 minutes at 4 C 12 visit us on the web at www qbiogene com 2niX Midiprep Kit 20 Completely remove ALL of the supernatant from the pellet with a pipette 21 Air dry the pellet for 10 minutes Note Drying with a vacuum chamber is not recommended because over dried DNA may be difficult to completely resuspend 22 Dissolve the plasmid DNA in 100 ul of water or TE Buffer DNA is immediately ready for downstream applications and may be moved to a smaller tube if desired 7 Troubleshooting 7 1 Low yields of plasmid DNA 7 1 1 Temperature was too low Temperature plays an important role in the isolation of optimal amounts of plasmid DNA All solutions should be kept no cooler than room temperature 18 25 C If cleared lysate was centrifuged at 4 C the super natant must be allowed to warm prior to adding to the column Wash and elution buffers must also be used at room temperature 7 1 2 Culture medium was not completely removed Plasmid DNA will not bind optimally to the PhoenIX Midi Column unless it is at the correct salt concen tration and pH Excess culture media that is resuspended with the cells in the Cell Resuspension B
24. will become irreversibly denatured if alkaline cell lysis step 7 continues longer than the recommended 5 minutes 7 5 RNA contamination 7 5 1 Preparation was at the incorrect salt concentration pH or temperature Due to the nature of the PhoenIX ion exchange resin the separation of DNA and RNA is strongly dependent on the salt concentration pH and temperature during binding washing and elution Follow the protocol exactly as directed and see sections 7 1 1 7 1 3 under Low yields of plasmid DNA 7 5 2 Sample was left on the column too long Once the cleared bacterial lysate is added to the column proceed with subsequent steps with no inter ruptions Long pauses between steps may cause contamination of the purified plasmid DNA with small RNA species visit us on the web at www qbiogene com 2niX Midiprep Kit 7 5 3 RNase A digestion was insufficient Ensure the appropriate amount of RNase A was added to the Cell Resuspension Buffer prior to beginning the protocol see section 4 3 If the Cell Resuspension Buffer was not stored at 4 C or if it has been stored at 4 C for more than 6 months new RNase A should be added prior to purifying more plasmid DNA Ensure the correct volume of prepared Cell Resuspension Buffer was added in step 5 of the protocol 7 5 4 Host strain may be naturally rich in RNA some host strains are extremely rich in RNA Therefore it is possible that a slight contamination of the plasmid
25. x 9 7 Add 4 mi of Lysis Buffer blue cap label and securely cap the tube Mix thoroughly by inverting until the lysate appears to be homogeneous 5 6 inversions DO NOT VORTEX 8 Incubate 5 minutes at room temperature Note Do not incubate for longer than 5 minutes or plasmid DNA might become irreversibly denatured 9 Add 4 ml of Neutralization Buffer green cap label Securely cap the tube and mix immediately by mul tiple inversions until a homogeneous suspension containing no viscous matter is obtained DO NOT VORTEX Note f preparing several samples at once thoroughly mix each sample immediately after the addition of the Neutralization Buffer before adding the buffer to the next tube 10 Centrifuge the thoroughly mixed sample at 12 000 x g for 10 minutes at room temperature Note f a room temperature centrifuge is not available and sample is centrifuged at 4 C the supernatant must be allowed to warm to room temperature 18 25 C prior to loading on the PhoenIX Midi Column 11 Verify that the Equilibration Buffer has been collected in the waste container beneath the PhoenIX Midi Column Discard the flow through and replace the container visit us on the web at www qbiogene com 12 Remove 480 ul of cleared lysate from step 10 and transfer it to a separate 1 5 ml microcentrifuge tube THIS IS DIAGNOSIS SAMPLE 1 13 Use a pipette to remove the remainder of the cleared lysate supernatant from the centrifu
26. y protocol to increase yield 7 1 6 Diagnosis protocol for low or no yield If the yield of an expected high copy plasmid still does not improve after the suggestions above are fol lowed perform the PhoenIX Midiprep Diagnosis Protocol in Appendix Il to determine if the problem lies with the supplied reagents or the procedure itself 7 2 Slow column flow If the flow of liquid through the PhoenIX Midi Column slows or stops it may be due to the clogging of the column with particulate matter carried over from the cleared lysate in step 12 Do not pour the cleared lysate onto the column right from the centrifuge tube Carefully separate the cleared lysate from the particulate matter with a pipet and load the column as directed in the protocol 7 3 Chromosomal DNA contamination Chromosomal bacterial DNA is removed from the preparation by precipitation after the addition of the Neutralization Buffer and by subsequent centrifugation This is only successful if shearing of the chromo somal DNA after cell lysis is kept to a minimum Shearing of the chromosomal DNA occurs when the sample is vortexed after the addition of Lysis Buffer and or Neutralization Buffer Mix only by inversion after the addi tion of these solutions do not vortex 7 4 Multiple plasmid forms seen on agarose gel A DNA band that runs slightly faster than the supercoiled plasmid DNA on a gel most likely represents irre versibly denatured plasmid DNA Plasmid DNA
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