Providing Marligen PowerPrep™ HP Plasmid Purification
Contents
1. 25 Reactions 50 reactions 10 Reactions 25 Reactions 4 Reactions 2 Reactions 25 Reactions 50 Reactions 10 Reactions 25 Reactions TABLE OF CONTENTS Notices to Customers isis sivssisecisntisauisesscantiscananisswasentdnienentiaasanddeesaanisavenatduunsessssiaenadaninansdinivens 2 OVE VIC ccssdiuiiisis cutdinenicatanicnd nests leanthinandsnabeusesidanscianabucasuidcasaannibuasanddusstiedaxsainandnasmadanaddaseerdensssans 2 Components and Storage iiiessisccissndsewananisinanvadnindvs caw onsnnndiniduaadadwneadindbuaaaieuanseiieiinnnanauananawodians 3 Critical Parameters and Protocol Notes cccccccessseeeseeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeseneeeeeeeeeeeeees 4 Methods HP Miniprep ProGedure esesseegueruegrueueseRSeueEEEEENEEENESERERSSEEEEEENEEENEEESENCNEEEEEEKEEENERERKSENEEEEEEN 6 HP Midiprep PrOCOQUi eG ivvcicsiacisiccsccecisscantstnasancedensecsstnarandscnasensuieasesananasaaceduecseanananancncaews 8 FIP Maxiprep ET EN 10 HP Midiprep Procedure with prefilters ccccccssssseseeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeneees 12 HP Maxiprep Procedure with prefilters ccccccsssssseeeeeeeeeeeeeeseeeeeeeeeeeeeeeeeeneeeeeeees 14 Quick Reference Protocol sisiccscsiscsiewsienccsecwnnssisouewecsxeuewnusntuasouceevenauswecevecacvenstiecewecnsts 16 Overview for Megaprep and Gigaprep Kits ccccccesseeeeeeeeeeeeeeeeeeeeeeseeeeeeeeeeeeeeeeeeeeeeeeees 17 Methods HP Meg aprep Procedure wivisccissieceiicenscaveieieraser2. ORIGENE NOTICE All purification kits with catalog numbers ending in NP1000xxN do not include any buffers A protocol detailing how to create necessary buffers is included with each purification kit ending in NP1000xxN Providing Marligen PowerPrep HP Plasmid Purification Systems For the isolation of HIGH PURITY plasmid cosmid and BAC DNA suitable for all molecular and cellular biology applications 9620 Medical Center Drive Suite 200 Rockville MD 20850 888 267 4436 phone 301 340 9254 fax POWERPREP PowerPrep HP Plasmid Miniprep Kits PowerPrep HP Plasmid Midiprep Kits PowerPrep HP Plasmid Maxiprep Kits PowerPrep HP Plasmid Megaprep Kits PowerPrep HP Plasmid Gigaprep Kits PowerPrep HP Plasmid Midiprep Kits with Prefilters PowerPrep HP Plasmid Maxiprep Kits with Prefilters Catalog No Catalog No Catalog No Catalog No Catalog No Catalog No Catalog No Catalog No Catalog No Catalog No Catalog No Catalog No 9620 Medical Center Drive Suite 200 Rockville MD 20850 888 267 4436 phone 301 340 9254 fax NP100004 11449 014 NP100005 11449 022 NP100006 11451 010 NP100007 11451 028 NP100008 11452 018 NP100009 11452 026 NP100020 11462 004 NP100021 11463 002 NP100022 11475 025 NP100023 11475 050 NP100024 11476 010 NP100025 11476 025 2 HP PLASMID PURIFICATION KITS 25 Reactions 100 Reactions
3. 150 mL receiver flasks are reusable if washed thoroughly and show no signs of wear or cracking In order to avoid cross contamination it is highly recommended that the receiver flasks only be re used for identical plasmid preps To avoid the possibility of implosion do not use any vessels that are not designed for use with vacuum Do not use bottles flasks or any other vessels that are cracked or scratched Always wear safety glasses when working near a boitle or flask under vacuum Purification of high purity endotoxin free DNA with the novel OriGene Mega and Giga Plasmid Kits is quite different than purification of DNA with the Mini Midi Maxi kits that are based on gravity flow columns New users are strongly advised to read the entire protocol very carefully before starting the procedure Although still based on a patented anion exchange chromatography the OriGene Megaprep and Gigaprep Kits are not based on gravity flow columns but consist of the high purity anion exchange resin packed in a vacuum driven filter cartridge Vacuum is applied to the cartridge by a conventional water jet filter pump or a vacuum pump The PowerPrep HP Megaprep Kit is appropriate for culture volumes of 500 mL to 2 5 liters The nominal capacity of the Megaprep DNA binding cartridges is 2 5 mg of DNA For high copy plasmids pTZ pBluescript pUC and other plasmids that are present in E coli cultures at concentrations of 4 5 ug mL we recommend the use of not more
4. mL Elution Buffer E Vv 3 5 mL om isopropanol 8 KN S RW mL 70 ethanol exw v smn NY 200uL TE Buffer 1 Column Equilibration Apply 10 mL of Equilibration Buffer to the column Allow the solution in the column to drain by gravity flow NOTE If using the filtration method for lysate clarification pour the Equilibration Buffer through the filter paper in the funnel that was placed in the column Cell Harvesting For high copy number plasmids gt 2 ug DNA mL culture pellet 15 to 25 mL of an overnight culture For low copy number plasmids 2 ug DNA mL culture pellet 25 to 100 mL of an overnight culture Thoroughly remove all medium Cell Suspension Add 4 mL of Cell Suspension Buffer containing RNase A to the pellet and suspend the cells until homogeneous Cell Lysis Add 4 mL of Cell Lysis Solution Mix gently by inverting the capped tube five times Do not vortex Incubate at room temperature for exactly 5 min Neutralization Add 4 mL of Neutralization Buffer and mix immediately by inverting the tube until the mixture is homogeneous When large cell pellets have been processed more vigorous shaking may be required However DO NOT VORTEX Centrifuge the mixture at 15 000 x g at room temperature for 10 min If centrifugation is done at 4 C supernatant must be warmed to room temperature before loading on column Column Loading Pipet the supernatant from step 5 onto the equilibrated colum
5. mL culture pellet 1 to 3 mL of an overnight culture For low copy number plasmids 2 ug DNA mL culture pellet 10 to 15 mL of an overnight culture Thoroughly remove all medium 3 Cell Suspension Add 0 4 mL of Cell Suspension Buffer containing RNase A to the pellet and suspend the cells until homogeneous 4 Cell Lysis Add 0 4 mL of Cell Lysis Solution Mix gently by inverting the capped tube five times Do not vortex Incubate at room temperature for exactly 5 min 5 Neutralization Add 0 4 mL 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 However DO NOT VORTEX Centrifuge the mixture at 12 000 x g at room temperature for 10 min If centrifugation is done at 4 C supernatant must be warmed to room temperature before loading on column 6 Column Loading Pipet the supernatant from step 5 onto the equilibrated column Allow the solution in the column to drain by gravity flow Discard flow through 7 Column Wash Wash the column two times with 2 5 mL of Wash Buffer Allow the solution in the column to drain by gravity flow after each wash Discard flow through 8 Plasmid DNA Elution Elute the DNA by adding 0 9 mL of Elution Buffer Allow the solution in the column to drain by gravity flow Do not force out remaining solution 9 Plasmid DNA Precipitation Add 0 63 mL of isopropanol to t
6. mL 30 mL 30 mL Cell Harvesting and Alkaline Lysis Cell Suspension Buffer 0 4 mL 4mL 10 mL 10 mL 10 mL Cell Lysis Buffer 0 4 mL 4 mL 10 mL 10 mL 10 mL Neutralization Buffer 0 4 mL 4 mL 10 mL 10 mL 10 mL Column Loading and Elution Wash Buffer 10 mL 10 mL Discard Discard Prefilter Prefilter Wash Buffer 2x2 5mL 2x10 mL 20 mL 60 mL 50 mL Elution Buffer 0 9 mL 5 mL 5 mL 15 mL 15 mL Isopropanol 0 63 mL 3 5 mL 3 5 mL 10 5 mL 10 5 mL 70 Ethanol 1 mL 3 mL 5 mL 5 mL 5 mL TE Buffer 50 uL 200 uL 200 ul 500 uL 500 uL Additional volumes may be required for Low Copy Number plasmids Cat No NP100003 Lysate Wash for kits with prefilters 9620 Medical Center Drive Suite 200 Rockville MD 20850 888 267 4436 phone 301 340 9254 fax MEGA and GIGAPREP OVERVIEW AND PROTOCOL NOTES Additional Materials Required for Mega and Gigapreps e A vacuum source capable of generating a negative pressure of 20 inches Hg 600 to 800 mbar It is essential that a vacuum control device be placed just next to the filter bottle so that the vacuum pressure can be adjusted down to 5 to 8 in Hg during the procedure e A 1000 mL Stericup Receiver flask with 45 mm thread Millipore Cat SCOO B10 RE also available from Fisher Scientific e A150 mL Stericup Receiver flask with 45 mm thread Millipore Cat No SCOO B01 RE also available from Fisher Scientific See http Awww millipore com catalogue nsf docs C3239 NOTE The 1000 mL and
7. medium 3 Cell Suspension Add 10 mL of Cell Suspension Buffer containing RNase A to the pellet and suspend the cells until homogeneous For culture volumes greater than 200 mL add 20 mL of Cell Suspension buffer 4 Cell Lysis Add 10 mL of Cell Lysis Solution For culture volumes greater than 200 mL add 20 mL of Cell Lysis Solution Mix gently by inverting the capped tube five times Do not vortex Incubate at room temperature for exactly 5 min 5 Neutralization Add 10 mL of Neutralization Buffer and mix immediately by inverting the tube until the solution is homogeneous For culture volumes greater than 200 mL add 20 mL 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 However DO NOT VORTEX 6 Column Loading Pour the neutralized cell lysate including all of the precipitated material into the previously equilibrated prefilter column combination Let the lysate run through by gravity flow until the flow stops or becomes very slow lt 1 drop per 10 seconds Discard flow through 7 Lysate Wash Add 10 mL of Wash Buffer to the prefilter and let drain by gravity until the flow stops or becomes very slow Do not force any remaining liquid out of the Prefilter Prefilter Removal As soon has the column as stopped dripping remove the prefilter from the column and discard it 9 Column Wash Wash th
8. min Carefully discard supernatant Wash the plasmid DNA pellet with 3 mL of 70 ethanol and centrifuge at 15 000 x g at 4 C for 5 min Carefully and fully pipet off the ethanol wash Air dry the pellet for 10 min NOTE The DNA pellet is easily dislodged when washing with 70 ethanol It is best to pipet off the ethanol wash to remove it from the pellet This is a particular problem when the DNA pellet is very small 12 Purified DNA Dissolve the pelleted DNA in 200 uL of TE Buffer Occasionally insoluble particles are present These particles do not influence the quality of DNA and can easily be removed by centrifugation in a microcentrifuge at 12 000 x g at room temperature for 1 min Transfer the sample to a fresh tube 9620 Medical Center Drive Suite 200 Rockville MD 20850 888 267 4436 phone 301 340 9254 fax MAXIPREP PROTOCOL WITH INTEGRATED PREFILTERS BEFORE BEGINNING Verify that RNase A has been added to Cell Suspension Buffer and that no precipitate has formed in Cell Lysis Solution See Advance Preparations 1 Column Equilibration Apply 30 mL of Equilibration Buffer directly into the prefilter that is inserted in the column Allow the solution in the column to drain by gravity flow 2 Cell Harvesting For high copy number plasmids gt 2 ug DNA mL culture pellet up to 100 mL of an overnight culture For low copy number plasmids 2 ug DNA mL culture pellet 200 to 500 mL of an overnight culture Thoroughly remove all
9. 0 20 mL of Elution Buffer have been pulled through the cartridge Release the vacuum from the cartridge so that no further liquid is pulled through the resin Let stand for 1 minute without agitation Then switch on the vacuum source again and draw the remaining liquid from the resin into the receiver bottle Keep the vacuum on until all liquid has drained from the resin NOTE The final DNA yield can be increased by approximately 10 if a second elution step with another 100 mL of Elution Buffer is carried out as described 9620 Medical Center Drive Suite 200 Rockville MD 20850 888 267 4436 phone 301 340 9254 fax 11 DNA Precipitation Precipitate the DNA with 0 7 volumes of isopropanol Centrifuge at 4 C and gt 12 000 x g for at least 30 minutes Wash the precipitated DNA with 20 mL of 70 80 ethanol per tube and re centrifuge for 5 minutes Air dry the DNA pellet for 10 minutes and redissolve it in a suitable volume of TE Buffer Note Plasmid DNA may spread over the whole wall of the centrifuge tube if a fixed angle rotor is used Therefore we suggest the use of a swing out rotor that allows centrifugal forces of gt 12 000 x g HB 4 or HB 6 for Sorvall centrifuges or if such a rotor is not available the siliconization of the centrifuge tubes with a repellent silane e dimethyldichlorosilane The DNA pellet is easily dislodged when washing with 70 ethanol It is best to pipet off the isopropanol supernatant and ethanol wash to re
10. 0 mL 250 mL Buffer Equilibration 50 mL 250 mL 250 mL 2 299 300 mL 2 4 400 mL 400 mL 400 mL 2 4 300 m 2 40 Buffer mL mL mL mL 2x 500 2x 300 3x500 4x400 3x 400 2x400 3x500 2x300 3x 500 Wash Buffer 125 mL 500 mL 500mL 74 in a sr Sg g ee Elution Buffer 25mL 125ml 125 mL 250 mL 250 mL 400 mL 400 mL 400 mL 130 mL 250 mL 250 mL 400 mL TE Buffer mt 15mL 15mL 30mL 30mL 30mL 30mL 30mL 15mL 15mL 15mL 30mL Columns 25 100 25 50 10 25 4each 2each 25 50 10 25 Cartridges Lysate Filtration 4 each Sech Cartridges NOTICE All purification kits with catalog numbers ending in NP1000xxN do not include any buffers A protocol detailing how to create necessary buffers is included with each purification kit ending in NP1000xxN 9620 Medical Center Drive Suite 200 Rockville MD 20850 888 267 4436 phone 301 340 9254 fax 5 ADDITIONAL MATERIALS and PREPARATIONS Additional Materials Required Advance Preparations e lsopropanol e Add RNase A to Cell Suspension Buffer e 70 ethanol according to the instructions on the label of the e Nucleic Acid Purification Rack bottle Mix well Place a mark on the label to f indicate that RNase A has been added then e Tube appropriate for pelleting and lysing cells store Cell Suspension Buffer at 4 C Tube appropriate in size for collecting and e Check Cell
11. Buffer Vv Harvest Cells Vv 10 mL Cell Suspension Buffer Vv 10 mL Cell Lysis Solution ECH A 10 mL Neutralization Buffer Vv Load Column Vv Dinge d itt me iit pg lt N LE N 60 mL Wash Buffer Vv 15 mL Elution Buffer Vv 10 5 mL isopropanol mm y 5 mL 70 ethanol d 500 uL TE Buffer MAXIPREP PROTOCOL BEFORE BEGINNING Verify that RNase A has been added to Cell Suspension Buffer and that no precipitate has formed in Cell Lysis Solution See Advance Preparations 1 Column Equilibration Apply 30 mL of Equilibration Buffer to the column Allow the solution in the column to drain by gravity flow NOTE If using the filtration method for lysate clarification pour the Equilibration Buffer through the filter paper in the funnel that was placed in the column 2 Cell Harvesting For high copy number plasmids gt 2 ug DNA mL culture pellet 100 mL of an overnight culture For low copy number plasmids 2 ug DNA mL culture pellet 250 to 500 mL of an overnight culture Thoroughly remove all medium 3 Cell Suspension Add 10 mL of Cell Suspension Buffer containing RNase A to the pellet and suspend the cells until homogeneous 4 Cell Lysis Add 10 mL of Cell Lysis Solution Mix gently by inverting the capped tube five times Do not vortex Incubate at room temperature for exactly 5 min 5 Neutralization Add 10 mL of Neutralization Buffer and mix immediatel
12. DNA DO NOT VORTEX Incubate at room temperature for exactly 5 min Neutralization Add 125 mL of Neutralization Buffer and mix immediately by inverting the tube at least five times until the solution is homogeneous A white flocculent precipitate made of proteins cellular debris genomic DNA and detergent will form When large cell pellets have been processed more vigorous shaking may be required However DO NOT VORTEX Lysate Filtration Pour the bacterial lysate from Step 5 directly into the prepared Gigaprep Lysate Filtration Cartridge from Step 1 Let stand at room temperature for at least 2 minutes without agitation Then attach a vacuum source to the tubing connector and apply vacuum Collect the clear flow through into the bottle Keep the vacuum on until all liquid has drained from the unit Then switch off the vacuum source IMPORTANT NOTE It is very important to let the lysate stand for at least 5 minutes after the transfer into the cartridge This allows the precipitate to float and form a layer on top of the lysate and ensures convenient filtration without clogging As a rule of thumb one can expect to recover up to 330 mL of filtrate Cleared Lysate Collection Add 50 mL of Wash Buffer to the Gigaprep Lysate Filtration Cartridge and gently stir the precipitate with a sterile spatula Connect the vacuum source again and apply vacuum until all liquid has been pulled through completely Gentle agitation of the precipitate improves the fl
13. Lysis Solution for precipitate If precipitating plasmid DNA eluted from column necessary warm the solution briefly at 37 C to e For minipreps microcentrifuge capable of reaching dissolve the precipitate 12 000 x g at room temperature and 4 C e Store all components at room temperature For midipreps and maxipreps centrifuge and rotor capable of reaching 15 000 x g at room temperature and 4 C For mega and gigapreps vacuum controller 1000 mL receiver flask 150 mL receiver flask See page14 for details CRITICAL PARAMETERS AND PROTOCOL NOTES General e For optimal performance use volumes temperatures incubation times and centrifugations precisely as indicated in the protocol e Store columns and solutions at recommended temperatures e Cultures may be grown in LB medium or rich media including Superbroth Terrific Broth 2XYT or other proprietary media Cell density should be 1 4 Asoo units mL e Do not overload the columns Use the recommended culture volumes as indicated in the protocol to obtain optimal yield and purity e Modified protocols for purifying DNA from BACs Bacmids Cosmids and M13 can be found on our website at www OriGene com or may be requested by calling customer service at 888 267 4436 Additional volumes of some buffers are required to purify DNA from BACs These buffers can be purchased separately as the PowerPrep HP BAC Buffer Kit Cat No NP100003 e lf you are processing more than 50 ml o
14. e column with 50 mL of Wash Buffer Allow the solution in the column to drain by gravity flow Discard flow through 10 Plasmid DNA Elution Elute the DNA by adding 15 mL of Elution Buffer Allow the solution in the column to drain by gravity flow Do not force out remaining solution 11 Plasmid DNA Precipitation Add 10 5 mL of isopropanol to the eluate Mix and centrifuge the mixture at 15 000 x g at 4 C for 30 min Carefully discard supernatant Wash the plasmid DNA pellet with 5 mL of 70 ethanol and centrifuge at 15 000 x g at 4 C for 5 min Carefully and fully pipet off the ethanol wash Air dry the pellet for 10 min NOTE The DNA pellet is easily dislodged when washing with 70 ethanol It is best to pipet off the ethanol wash to remove it from the pellet This is a particular problem when the DNA pellet is very small 12 Purified DNA Dissolve the pelleted DNA in 500 uL of TE Buffer Occasionally insoluble particles are present These particles do not influence the quality of DNA and can easily be removed by centrifugation in a microcentrifuge at 12 000 x g at room temperature for 1 min Transfer the sample to a fresh tube 9620 Medical Center Drive Suite 200 Rockville MD 20850 888 267 4436 phone 301 340 9254 fax Quick Reference Protocol Volumes Per Reaction Miniprep Midiprep VE Ge Maxiprep oe Column Equilibration Equilibration Buffer 2mL 10 mL 15
15. eactions NP100014 For additional product information protocols and troubleshooting information visit our website at www OriGene com Contact Information OriGene Technologies Inc 9620 Medical Center Drive Suite 200 Rockville MD 20850 custsupport OriGene com techsupport OriGene com 9620 Medical Center Drive Suite 200 Rockville MD 20850 888 267 4436 phone 301 340 9254 fax
16. elleted DNA in 500 uL of TE Buffer Occasionally insoluble particles are present These particles do not influence the quality of DNA and can easily be removed by centrifugation in a microcentrifuge at 12 000 x g at room temperature for 1 min Transfer the sample to a fresh tube 9620 Medical Center Drive Suite 200 Rockville MD 20850 888 267 4436 phone 301 340 9254 fax MIDIPREP PROTOCOL WITH INTEGRATED PREFILTERS BEFORE BEGINNING Verify that RNase A has been added to Cell Suspension Buffer and that no precipitate has formed in Cell Lysis Solution See Advance Preparations 1 Column Equilibration Apply 15 mL of Equilibration Buffer directly into the prefilter that is inserted in the column Allow the solution in the column to drain by gravity flow Prepare cell lysate while the column and prefilter unit are equilibrating Note Shortly after the column has begun dripping some drops at the outlet may appear turbid This is normal and due to the interaction of the equilibration buffer with the resin The turbid drops will not affect the preparation in any way 2 Cell Harvesting For high copy number plasmids gt 2 ug DNA mL culture pellet up to 50 mL of an overnight culture For low copy number plasmids 2 ug DNA mL culture pellet 50 to 100 mL of an overnight culture Thoroughly remove all medium 3 Cell Suspension Add 10 mL of Cell Suspension Buffer containing RNase A to the pellet and suspend the cells until the mixtu
17. erewecsvenesararaniwaweverecerisanenesevenecerereneranacusion 18 HP Gigaprep Procedure ss sisisinsndnisstwawivininendnenawsnauesinaninnadwaninla niweninaninaaduesduauilesinaniuandsand 20 Uebst TE 22 Accessories and Related Products seiscscscsssisssssctsecccssnsssansstannanscsdssecssseedansanuacesssacstaneaauancaass 23 9620 Medical Center Drive Suite 200 Rockville MD 20850 888 267 4436 phone 301 340 9254 fax 3 NOTICES TO CUSTOMER Important Information The product you have received is authorized for laboratory research use only The product has not been qualified or found safe and effective for any human or animal diagnostic or therapeutic application Uses other than the labeled intended use may be a violation of applicable law Precautions Warning 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 OVERVIEW The PowerPrep HP Plasmid Purification Kits use a unique anion exchange resin to purify plasmid DNA to a level equivalent to two passes through CsCl gradients The kits with prefilters add the convenience of an integrated filter unit that allows simultaneous one step lysate clarifica
18. f culture for Midiprep columns or more than 200 ml for Maxiprep columns to purify low copy plasmids we strongly recommend that you double the standard volumes of Cell Suspension Buffer with double the RNase concentration Lysis Buffer and Neutralization Buffer used for preparation of the cell lysate The buffer volumes included in the kit however are only sufficient for the designed number of standard preps Therefore additional buffer is required in order to utilize all of the columns in the kit for processing large culture volumes These buffers can be purchased separately as the PowerPrep HP BAC Buffer Kit Cat No NP100003 9620 Medical Center Drive Suite 200 Rockville MD 20850 888 267 4436 phone 301 340 9254 fax 6 Important Considerations for Alkaline Lysis and Neutralization Steps Suspension For efficient lysis it is important to use a vessel that is large enough to allow complete mixing of the lysis buffers Ensure 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 Lysis Mix gently but thoroughly Do not vortex as this may result in shearing of genomic DNA and contamination of the plasmid DNA The lysate should appear viscous Do not allow the lysis reaction to proceed for more than 5 min Neutralization After addition of Neutralization Buffer a fluffy white material should form and the lysate should beco
19. he eluate Mix and centrifuge the mixture at 12 000 x g at 4 C for 30 min Carefully discard supernatant Wash the plasmid DNA pellet with 1 mL of 70 ethanol and centrifuge at 12 000 x g at 4 C for 5 min Carefully and fully pipet off the ethanol wash Air dry the pellet for 10 min NOTE The DNA pellet is easily dislodged when washing with 70 ethanol It is best to pipet off the isopropanol supernatant and ethanol wash to remove these liquids from the pellet This is a particular problem for minipreps or when the DNA pellet is very small 10 Purified DNA Dissolve the pelleted DNA in 50 uL of TE Buffer Occasionally insoluble particles are present These particles do not influence the quality of DNA and can easily be removed by centrifugation at 12 000 x g at room temperature for 1 min Transfer the sample to a fresh tube 9620 Medical Center Drive Suite 200 Rockville MD 20850 888 267 4436 phone 301 340 9254 fax MIDIPREP PROTOCOL BEFORE BEGINNING Verify that RNase A has been added to Cell Suspension Buffer and that no precipitate has formed in Cell Lysis Solution See Advance Preparations 10 mL Equilibration Buffer Vv C Harvest Cells Vv 4 mL Cell Suspension Buffer j H d MN TTT Vv mg M 4 mL Cell Lysis Solution VN 5MIN A MTT 4 mL Neutralization Buffer H Wu yT a Vv mmm j Load Column Vv MM 2x10 mL Wash Buffer I Vv 5
20. igh quality and is suitable for all molecular and cell biology applications including transfection automated fluorescent DNA sequencing manual DNA sequencing amplification reactions in vitro transcription cloning and labeling Reference 1 Birnboim H and Doly J 1979 Nucleic Acids Res 7 1513 This product is the subject of U S Patent No 5 843 312 and foreign equivalents 9620 Medical Center Drive Suite 200 Rockville MD 20850 888 267 4436 phone 301 340 9254 fax 4 COMPONENTS Store all components at room temperature After the addition of RNase A to the Cell Suspension Buffer store at 4 C NOTE The Megaprep kit is contained in two separate boxes a cartridge box and a solutions box VOLUMES OF COMPONENTS MINIPREP MIDIPREP MAXIPREP MEGA GIGA MIDIPREP KIT MAXIPREP KITS KITS KITS WITH KITS WITH PREFILTERS PREFILTERS COMPONENT 25 RXN 100 RXN 25 RXN 50 RXN 10 RXN 25 RXN 4RXN 2RXN 25 RXN 50 RXN 10 RXN 25 RXN NAME Cell Suspension Buffer 15 mL 65mL 100 mL 250 mL 100 mL 250 mL 250 mL 250 mL 250 mL 500 mL 100 mL 250 mL RNase A 100 uL 650 uL 650 uL 1 5mL 650 uL 1 5mL 1 5mL 1 5mL 1 5 mL 2 8 mL 650 ul 1 5 mL Cell Lysis Solution 15 mL 65 mL 100 mL 250 mL 100 mL 250 mL 250 mL 250 mL 250 mL 500 mL 100 mL 250 mL Neutralization 10mL 40mL 100mL 200mL 100 mL 250 mL 200 mL 250mL 250 mL 500 mL 10
21. ll centrifuges or if such a rotor is not available the siliconization of the centrifuge tubes with a repellent silane e dimethyldichlorosilane The DNA pellet is easily dislodged when washing with 70 ethanol It is best to pipet off the isopropanol supernatant and ethanol wash to remove these liquids from the pellet 9620 Medical Center Drive Suite 200 Rockville MD 20850 888 267 4436 phone 301 340 9254 fax GIGAPREP PROTOCOL BEFORE BEGINNING Verify that RNase A has been added to Cell Suspension Buffer and that no precipitate has formed in Cell Lysis Solution See Advance Preparations 1 10 Setup Screw the Gigaprep Lysate Filtration Cartridge onto a clean 1000 mL Stericup Receiver flask NOTE Do not overtighten the filtration cartridge on the bottle neck since the filtration cartridge plastic may crack Cell Harvesting For high copy number plasmids gt 2 ug DNA mL culture pellet 2 5 L of an overnight culture For low copy number plasmids 2 ug DNA mL culture pellet 2 5 to 5 L of an overnight culture Thoroughly remove all medium Cell Suspension Add 125 mL of Cell Suspension Buffer containing RNase A to the pellet and suspend the cells until homogeneous Be sure that no cell clumps are visible Cell Lysis Add 125 mL of Cell Lysis Solution Mix gently but thoroughly by inverting at least five times until a homogeneous solution is obtained The mixture is very viscous at this stage due to the release of genomic
22. me 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 Protocol for Clearing Lysates by Filtration for Midi and Maxipreps not necessary if using kits with prefilters Catalog s NP100022 NP100023 NP100024 NP100025 This protocol modification provides a fast and inexpensive method for clarification of cell lysates for the Midi and Maxi size preps The Mega and Gigapreps use a vacuum assisted filter cartridge to clarify cell lysates Plasmid DNA prepared with this method is generally of higher purity than plasmid DNA obtained from lysates cleared by centrifugation because the precipitate is completely removed and does not enter the column Recommended Materials Funnel Filter Paper Tyco Healthcare 65 mm Whatman 2V fluted filter Midiprep Polystyrene Funnel 8889 paper 216100 125 mm 1202 125 Maxipre Nalgene 80 mm Polypropylene Whatman 2V fluted filter prep Powder Funnel 4252 0080 paper 150 mm 1202 150 i Before beginning place the funnel in the top of the column and insert the fluted Whatman 2V filter paper into the funnel Apply the entire required amount of Equilibration Buffer to the filter and allow the solu
23. move these liquids from the pellet 9620 Medical Center Drive Suite 200 Rockville MD 20850 888 267 4436 phone 301 340 9254 fax TROUBLESHOOTING GUIDE Problem Possible Cause Suggested Solution Low yield of plasmid DNA Temperature of buffers too Store all buffers except Cell Suspension Buffer low with RNAse A at room temperature Lysate centrifuged at 4 C Ensure that rotor and centrifuge are at room temperature for lysate centrifugation step Low copy number plasmid Increase the number of cells processed Lysate at improper pH or Carefully remove all medium before suspending salt concentration cells Plasmid DNA pellet over Air dry the plasmid DNA pellet so that it will fully dried dissolve Do not dry the pellet with a vacuum system DNA pellet lost during It is easy to lose the DNA pellet during the ethanol wash ethanol wash especially for the miniprep size Pipet the ethanol from the tube prior to drying Slow column flow Column clogged Pipette lysate supernatant onto column Pouring Mini Midi Maxi lysate can result in precipitate particles entering the column Chromosomal DNA Genomic DNA sheared in Invert tubes when adding Cell Lysis and contamination handling Neutralization Buffers Do not vortex Additional plasmid forms Plasmid DNA permanently Incubate the lysate at room temperature for a present denatured maximum of 5 minutes Permanently denatured DNA will appear as a band electrophoresing just ahead of the superc
24. n Allow the solution in the column to drain by gravity flow Discard flow through Column Wash Wash the column two times with 10 mL of Wash Buffer Allow the solution in the column to drain by gravity flow after each wash Discard flow through Plasmid DNA Elution Elute the DNA by adding 5 mL of Elution Buffer Allow the solution in the column to drain by gravity flow Do not force out remaining solution Plasmid DNA Precipitation Add 3 5 mL of isopropanol to the eluate Mix and centrifuge the mixture at 15 000 x g at 4 C for 30 minutes Carefully discard supernatant Wash the plasmid DNA pellet with 3 mL of 70 ethanol and centrifuge at 15 000 x g at 4 C for 5 min Carefully and fully pipet off the ethanol wash Air dry the pellet for 10 min NOTE The DNA pellet is easily dislodged when washing with 70 ethanol It is best to pipet off the isopropanol supernatant and ethanol wash to remove these liquids from the pellet This is a particular problem for minipreps or when the DNA pellet is very small Purified DNA Dissolve the pelleted DNA in 200 uL of TE Buffer Occasionally insoluble particles are present These particles do not influence the quality of DNA and can easily be removed by centrifugation in a microcentrifuge at 12 000 x g at room temperature for 1 min Transfer the sample to a fresh tube 9620 Medical Center Drive Suite 200 Rockville MD 20850 888 267 4436 phone 301 340 9254 fax 30 mL Equilibration
25. o the pellet and suspend the cells until homogeneous Be sure that no cell clumps are visible Cell Lysis Add 50 mL of Cell Lysis Solution Mix gently but thoroughly by inverting at least five times until a homogeneous solution is obtained The mixture is very viscous at this stage due to the release of genomic DNA DO NOT VORTEX Incubate at room temperature for exactly 5 min Neutralization Add 50 mL of Neutralization Buffer and mix immediately by inverting the tube until the solution is homogeneous A white flocculent precipitate made of proteins cellular debris genomic DNA and detergent will form When large cell pellets have been processed more vigorous shaking may be required However DO NOT VORTEX Lysate Filtration Pour the bacterial lysate from Step 5 directly into the prepared Megaprep Lysate Filtration Cartridge from Step 1 Let stand at room temperature for at least 2 minutes without agitation Then attach a vacuum source to the tubing connector and apply vacuum Collect the clear flow through into the bottle Keep the vacuum on until all liquid has drained from the unit Then switch off the vacuum source IMPORTANT NOTE It is very important to let the lysate stand for at least 5 minutes after the transfer into the cartridge This allows the precipitate to float and form a layer on top of the lysate and ensures convenient filtration without clogging As a rule of thumb one can expect to recover up to 125 mL of filtrate Cleared Ly
26. oiled plasmid DNA This material will not be digested by restriction endonucleases RNA contamination Lysate at improper pH salt Carefully remove all medium before suspending concentration or cells temperature tor binding te Ensure that excess Neutralization Buffer is not column SS added when neutralizing the lysate Ensure that the lysate has not warmed above room temperature during the centrifugation Sample left on column too Once the lysate has been loaded on the column long or cartridge avoid delays in processing Lysate droplets remaining Wash droplets of lysate from walls of column or on walls of column at elution cartridge when adding wash buffer RNase A digestion Use recommended volume of Cell Suspension incomplete Buffer Ensure that Cell Suspension Buffer with RNase A is stored at 4 C and is less than 6 months old 9620 Medical Center Drive Suite 200 Rockville MD 20850 888 267 4436 phone 301 340 9254 fax ACCESSORIES SIZE CAT NO PowerPrep HP BAC Buffer Kit Each NP100003 RELATED PRODUCTS SIZE CAT NO PowerPrep Express Plasmid Miniprep Kits 50 reactions NP100010 250 reactions NP100011 PowerPrep Express PCR Purification Kits 50 reactions NP100015 250 reactions NP100016 PowerPrep Express 96 PCR Purification Kits 4 x 96 reactions NP100018 12 x 96 reactions NP100019 PowerPrep Express Gel Extraction Kits 50 reactions NP100012 250 reactions NP100013 PowerPrep Matrix Gel Extraction Kit 150 r
27. om the 1000 mL receiver flask and screw it onto a clean sterile 150 mL Stericup Receiver flask Add 50 mL of Elution Buffer into the Megaprep DNA Binding Cartridge Apply a soft vacuum 5 to 8 in Hg to the cartridge through the side arm with the tubing connector until approximately 10 20 mL of Elution Buffer have been pulled through the cartridge Release the vacuum from the cartridge so that no further liquid is pulled through the resin Let stand for 1 minute without agitation Then switch on the vacuum source again and draw the remaining liquid from the resin into the receiver bottle Keep the vacuum on until all liquid has drained from the resin NOTE The final DNA yield can be increased by approximately 10 if a second elution step with another 50 mL of Elution Buffer is carried out as described 9620 Medical Center Drive Suite 200 Rockville MD 20850 888 267 4436 phone 301 340 9254 fax 11 DNA Precipitation Precipitate the DNA with 0 7 volumes of isopropanol Centrifuge at 4 C and gt 12 000 x g for at least 30 minutes Wash the precipitated DNA with 20 mL of 70 80 ethanol per tube and re centrifuge for 5 minutes Air dry the DNA pellet for 10 minutes and redissolve it in a suitable volume of TE Buffer NOTE Plasmid DNA may spread over the whole wall of the centrifuge tube if a fixed angle rotor is used Therefore we suggest the use of a swing out rotor that allows centrifugal forces of gt 12 000 x g HB 4 or HB 6 for Sorva
28. ow of liquid through the filter unit The bottle now contains the filtered lysate containing the plasmid DNA Remove the Gigaprep Lysate Filtration Cartridge from the 1000 mL Stericup Receiver flask and decant the cleared lysate into a new sterile container DNA Binding Screw the Gigaprep DNA Binding Cartridge containing the ion exchange resin onto the 1000 mL Stericup Receiver flask and add 200 mL of Equilibration Buffer Apply vacuum to the cartridge through the side arm with the tubing connector and keep the vacuum on until all liquid has drained from the resin Discard the flow through Add the filtered cleared lysate from Step 7 into the Gigaprep DNA Binding Cartridge with the equilibrated resin and apply vacuum to the cartridge through the side arm with the tubing connector Keep the vacuum on until all of the lysate has passed through the resin Column Wash Add 275 mL of Wash Buffer into the cartridge and apply vacuum to the cartridge through the side arm with the tubing connector Repeat the wash with an additional 275 mL of Wash Buffer Keep the vacuum on until all liquid has drained from the resin DNA Elution Remove the Gigaprep DNA Binding Cartridge from the 1000 mL receiver flask and screw it onto a clean sterile 150 mL Stericup Receiver flask Add 100 mL of Elution Buffer into the Gigaprep DNA Binding Cartridge Apply a soft vacuum 5 to 8 in Hg to the cartridge through the side arm with the tubing connector until approximately 1
29. re is homogeneous 4 Cell Lysis Add 10 mL of Cell Lysis Solution and mix gently by inverting the capped tube five times Do not vortex Incubate at room temperature for exactly 5 min 5 Neutralization Add 10 mL 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 However DO NOT VORTEX 6 Column Loading Pour the neutralized cell lysate including all of the precipitated material into the previously equilibrated prefilter column combination Let the lysate run through by gravity flow until the flow stops or becomes very slow lt 1 drop per 10 seconds Discard flow through 7 Lysate Wash Add 10 mL of Wash Buffer to the prefilter and let drain by gravity until the flow stops or becomes very slow Do not force any remaining liquid out of the Prefilter 8 Prefilter Removal As soon as the column has stopped dripping remove the prefilter from the column and discard it 9 Column Wash Wash the column with 20 mL of Wash Buffer Allow the solution in the column to drain by gravity flow Discard flow through 10 Plasmid DNA Elution Elute the DNA by adding 5 mL of Elution Buffer Allow the solution in the column to drain by gravity flow Do not force out remaining solution 11 Plasmid DNA Precipitation Add 3 5 mL of isopropanol to the eluate Mix and centrifuge the mixture at 15 000 x g at 4 C for 30
30. sate Collection Add 50 mL of Wash Buffer to the Megaprep Lysate Filtration Cartridge and gently stir the precipitate with a sterile spatula Connect the vacuum source again and apply vacuum until all liquid has been pulled through completely Gentle agitation of the precipitate improves the flow of liquid through the filter unit The bottle now contains the filtered lysate containing the plasmid DNA Remove the Megaprep Lysate Filtration Cartridge from the 1000 mL Stericup Receiver flask and decant the cleared lysate into a new sterile container DNA Binding Screw the Megaprep DNA Binding Cartridge containing the ion exchange resin onto the 1000 mL Stericup Receiver flask and add 100 mL of Equilibration Buffer Apply vacuum to the cartridge through the side arm with the tubing connector and keep the vacuum on until all liquid has drained from the resin Discard the flow through Add the filtered cleared lysate from Step 7 into the Megaprep DNA Binding Cartridge with the equilibrated resin and apply vacuum to the cartridge through the side arm with the tubing connector Keep the vacuum on until all of the lysate has passed through the resin Column Wash Add 175 mL of Wash Buffer into the cartridge and apply vacuum to the cartridge through the side arm with the tubing connector Repeat the wash with an additional 175 mL of Wash Buffer Keep the vacuum on until all liquid has drained from the resin DNA Elution Remove the Megaprep DNA Binding Cartridge fr
31. than 500 mL of culture The maximum volume of 2 5 liters should only be used with low copy number plasmids cosmids or BACs The PowerPrep HP Gigaprep Kit is appropriate for culture volumes of 2 5 to 5 liters The nominal capacity of the Gigaprep DNA binding cartridges is 10 mg of DNA For high copy plasmids eg pTZ pBluescript pUC we recommend the use of not more than 2 5 liters of culture The maximum volume of 5 liters should only be used with low copy number plasmids or cosmids Critical Parameters Review all of the critical parameters on page 6 especially the notes regarding alkaline lysis and neutralization 9620 Medical Center Drive Suite 200 Rockville MD 20850 888 267 4436 phone 301 340 9254 fax MEGAPREP PROTOCOL BEFORE BEGINNING Verify that RNase A has been added to Cell Suspension Buffer and that no precipitate has formed in Cell Lysis Solution See Advance Preparations 1 10 Setup Screw the Megaprep Lysate Filtration Cartridge onto a clean 1000 mL Stericup Receiver flask NOTE Do not overtighten the filtration cartridge on the bottle neck since the filtration cartridge plastic may crack Cell Harvesting For high copy number plasmids gt 2 ug DNA mL culture pellet 500 mL of an overnight culture For low copy number plasmids 2 ug DNA mL culture pellet 500 mL to 2 5 L of an overnight culture Thoroughly remove all medium Cell Suspension Add 50 mL of Cell Suspension Buffer containing RNase A t
32. tion and column loading After a modified alkaline SDS procedure to lyse the cells and precipitate the genomic DNA the cleared lysate is passed over a pre packed ion exchange column 1 The negatively charged phosphates on the backbone of the DNA interact with the positive charges on the surface of the resin The temperature salt concentration and pH of the solutions influence binding Under moderate salt conditions plasmid DNA remains bound to the resin while RNA proteins carbohydrates and other impurities are washed off The plasmid DNA is eluted under high salt conditions It is then desalted and concentrated by alcohol precipitation The protocol can be completed in 1 5 to 2 hours These products eliminate the use of hazardous chemicals such as phenol chloroform and ethidium bromide e All types and sizes of plasmid DNA can be purified with the PowerPrep HP Plasmid Purification Kits Please contact Customer Service at custsupport origene com or at 888 267 4436 for modified protocols for BAC bacmid and ssM13 DNAs or visit our web site at www origene com e Yields of up to 30 ug miniprep 150 ug midiprep 750 ug maxiprep 2 5 mg megaprep and 10 mg gigaprep can be obtained with these systems Results are dependent on plasmid copy number plasmid type bacterial strain and growth conditions e g medium antibiotics temperature and aeration e Plasmid DNA purified using the PowerPrep HP Plasmid Purification Kits is of very h
33. tion to drain by gravity through the filter and column During equilibration proceed with the lysis and neutralization steps 2 After equilibration and neutralization pour the lysate into the filter The cleared lysate will flow through the filter and into the column The last 1 2 mL of cleared lysate can be recovered by gathering the top of the filter with gloved fingers and twisting gently When the lysate has finished flowing through the column remove the funnel containing the filter paper and discard the filter paper The funnel may be reused if desired Continue with the Column Wash step 7 9620 Medical Center Drive Suite 200 Rockville MD 20850 888 267 4436 phone 301 340 9254 fax MINIPREP PROTOCOL 2mL Equilibration Buffer NY Harvest Cells v 0 4 mL Cell Suspension Buffer v 0 4 mL JN Cell Lysis Solution smin KS NY 0 4 mL Neutralization Buffer La wi H Load Column Y 2x2 5mL Wash Buffer y NY SI 0 9 mL Elution Buffer y se pea A 0 63 mL Si l isopropanol E Vv 1 mL 70 ethanol Sg NV 50 uL TE Buffer BEFORE BEGINNING Verify that RNase A has been added to Cell Suspension Buffer and that no precipitate has formed in Cell Lysis Solution See Advance Preparations 1 Column Equilibration Apply 2 mL of Equilibration Buffer to the column Allow the solution in the column to drain by gravity flow 2 Cell Harvesting For high copy number plasmids gt 2 ug DNA
34. y by inverting the tube until the solution is homogeneous When large cell pellets have been processed more vigorous shaking may be required However DO NOT VORTEX Centrifuge the mixture at 15 000 x g at room temperature for 10 min If centrifugation is done at 4 C supernatant must be warmed to room temperature before loading on column 6 Column Loading Pipet the supernatant from step 5 onto the equilibrated column Allow the solution in the column to drain by gravity flow Discard flow through 7 Column Wash Wash the column with 60 mL of Wash Buffer Allow the solution in the column to drain by gravity flow Discard flow through 8 Plasmid DNA Elution Elute the DNA by adding 15 mL of Elution Buffer Allow the solution in the column to drain by gravity flow Do not force out remaining solution 9 Plasmid DNA Precipitation Add 10 5 mL of isopropanol to the eluate Mix and centrifuge the mixture at 15 000 x g at 4 C for 30 min Carefully discard supernatant Wash the plasmid DNA pellet with 5 mL of 70 ethanol and centrifuge at 15 000 x g at 4 C for 5 min Carefully and fully pipet off the ethanol wash Air dry the pellet for 10 min NOTE The DNA pellet is easily dislodged when washing with 70 ethanol It is best to pipet off the isopropanol supernatant and ethanol wash to remove these liquids from the pellet This is a particular problem for minipreps or when the DNA pellet is very small 10 Purified DNA Dissolve the p
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