Invisorb Spin Plasmid Mini Two User Manual
Contents
1. E mail info berlin stratec com www stratec com 1A101 10 20152. Important points before starting a protocol Immediately upon receipt of the Product inspect the Product and its components as well as the package for any apparent damages correct quantities and quality If there are any unconformities you have to notify STRATEC Molecular in writing with immediate effect upon inspection thereof If buffer bottles are damaged contact the STRATEC Molecular Technical Services or your local distributor In case of liquid spillage refer to Safety Information see page 5 Do not use damaged kit components since their use may lead to poor kit performance o Always change pipet tips between liquid transfers To avoid cross contamination we recommend the use of aerosol barrier pipet tips o All centrifugation steps should be carried out at room temperature o When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles o Discard gloves if they become contaminated o Do not mix kit components with components from other kits unless the lot numbers are identical o Avoid microbial contamination of the kit reagents Preparing reagents and buffers When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information please consult the appropriate material safety data sheets MSDS See at our webpage www stratec com 1 Label the needed amount of 2 0 ml receiver tubes per sample 2 receiver tubes 2 Place
3. Please note that a number of slightly different LB culture broths containing different concentrations of NaCl are in common use Although different LB broths produce similar cell densities after overnight culture plasmid yields may vary significantly 9 Invisorb Spin Plasmid Mini Two 1015 Scheme of Invisorb Spin Plasmid Mini Two Please read protocols prior the start of the preparation Transfer 0 5 ml up to 2 ml of the overnight E coli culture into a 1 5 ml or 2 0 ml microcentrifuge tube Centrifuge for 1 min at maximum speed 11 000 16 000 x g 11 000 13 000 rpm Remove the supernatant completely Resuspend the bacterial pellet in 250 ul Solution A Add 250 ul Solution B mix gently but thoroughly 4 6 times Add 250 ul Solution C mix gently by inverting the tube Centrifuge for 5 min at full speed 12 000 16 000 x g 11 000 13 000 rpm Transfer the clarified supernatant onto the Spin Filter Incubate for 1 min at RT Centrifuge for 1 min at 11 000 x g 11 000 rpm Discard the filtrate Add 750 ul Wash Solution Centrifuge for 1 min at 11 000 x g 11 000 rpm Discard the filtrate Centrifuge for 3 min at full speed 12 000 16 000 x g 11 000 13 000 rpm Place the Spin Filter into a new 1 5 ml Receiver Tube Add 50 100 ul Elution Solution Incubate for 1 min at room temperature Centrifuge at 11 000 x g 11 000 rpm for 1 min 10 Invisorb Spin Plasmid Mini Two 1015 Important notes
4. Storage of pDNA Store pDNA and other small circular DNAs at 2 to 8 C Storing pDNA at 15 to 25 C can cause shearing of DNA particularly if the DNA is exposed to repeated freeze thaw cycles 15 Invisorb Spin Plasmid Mini Two 1015 Examples of Plasmids Media Antibiotics and Bacterial strains Common Plasmid Vectors iti tie area and Copy Number Origin of Replication High Copy pBR322 15 20 PMB1 pUC 500 700 PMB1 mut pACYC 10 12 P15A pSC101 5 PSC101 pGEM 300 400 PMB1 mut pbluescript 300 500 ColE1 pTOPO n d PMB1 mut Common Media for bacterial culture 1L H medium TB terrific broth 10 g tryptone 12 g Bacto tryptone 8 g NaCl 24 g Ba to yeast extract 4 ml glycerol Lambda broth Add H2O to 900 ml and autoclave then add 10 g tryptone to above sterile solution 100 ml of a sterile 2 5 g NaCl solution of 0 17 M KH2PQ and 0 72 M K HPO4 LB medium 10 g tryptone Tryptone broth 5 g yeast extract 10 g tryptone 10g Naci 5g NaCl SOC Medium 2YT medium 20 g Bacto tryptone 16 g tryptone 5 g yeast extract 10 g yeast extract 100 mM Sodium chloride 5 g NaCl 2 5 mM Pottasium chloride 10 mM Magnesium chloride 20 mM Magnesium sulfate 20 mM Glucose Common Antibiotics Antibiotic Stock Solution Dissolved in Working Concentration Ampicillin 50 mg ml H2O 20 60 ug ml Carbenicillin 50 mg ml H2O 20 60 ug ml Chloramphenicol 34 mg ml H20 25 175
5. all buffers is optimized to provide high yield and purity of the isolated plasmid DNA The hands on time necessary for the whole procedure is reduced to a minimum Plasmid DNA purified by the Invisorb Spin Plasmid Mini Two is ready to use for a broad panel of downstream applications THE PRODUCT IS INTENDED FOR USE BY PROFESSIONALS ONLY SUCH AS TECHNICIANS PHYSICIANS AND BIOLOGISTS TRAINED IN MOLECULAR BIOLOGICAL TECHNIQUES It is designed to be used with any downstream application employing enzymatic amplification or other enzymatic modifications of RNA followed by signal detection or amplification 4 Invisorb Spin Plasmid Mini Two 1015 Any diagnostic results generated by using the sample preparation procedure in conjunction with any downstream diagnostic assay should be interpreted with regard to other clinical or laboratory findings To minimize irregularities in diagnostic results adequate controls for downstream applications should be used Product use limitation The Kit is neither for isolation and purification of DNA fragments or genomic DNA nor for isolation and purification of RNA The included chemicals are only useable once Differing of starting material or flow trace may lead to inoperability therefore neither a warranty nor guarantee in this case will be given neither implied nor express The user is responsible to validate the performance of the STRATEC Molecular Product for any particular use STRATEC Mo
6. in the clear supernatant Vigorous treatment during the lysis procedure will shear the bacterial chromosome leaving free chromosomal DNA fragments in the supernatant Since chromosomal fragments are chemically indistinguishable from plasmid DNA under the conditions used the two species will not be separated on Spin Filter membrane and will elute under the same low salt conditions Mixing during the lysis procedure must therefore be carried out by slow gentle inversion of the tube Binding plasmid DNA After cleaning of the supernatant each lysate is applied to an Invisorb Spin column and plasmid DNA is adsorbed onto membrane while the digested RNA cellular proteins and metabolites remain in the lysate and are drawn through by centrifugal force Removing residual contaminants Contaminations like endonucleases or others are efficiently washed away using Wash Solution while the plasmid DNA remains bound to the membrane Attention This washing step is essential when large cultured volumes are used or when working with endA strains such as the JM series HB101 and its derivates or any wild type strain to ensure that plasmid DNA is not degraded 7 Invisorb Spin Plasmid Mini Two 1015 Elution of plasmid DNA Plasmid DNA is eluted from the column using 50 100 ul Elution Solution Eluting twice each with 100 ul results in a slightly higher yield of DNA By the use of small elution volumes DNA concentration can be raised Elution volumes
7. should not fall below 50 ul otherwise the yield will be reduced The eluted DNA is ready to use in different downstream applications Note The Elution Solution contains EDTA Elution efficiency depends on pH The maximum elution efficiency is achieved between pH 7 0 8 5 When using water for elution make sure that the pH value is within this range Store pDNA frozen at 20 C when eluted with water since pDNA may degrade in the absence of a buffering agent DNA yield Plasmid yield with Invisorb Spin Plasmid Mini Two procedure varies depending on plasmid copy number per cell see page 19 the individual insert in a plasmid factors that affect growth of the bacterial culture see page 19 the elution volume and the elution incubation time A 2 0 ml overnight culture can yield 5 20 ug of plasmid DNA To obtain the optimum combination of DNA quality yield and concentration it is recommend to use an LB medium for growth of cultures using elution volume of 50 ul and performing incubation 1 3 min after addition of the Elution Solution Growth of bacterial cultures Plasmids are generally prepared from bacterial cultures grown in the presence of a selective agent such as an antibiotic The yield and quality of plasmid DNA may depend on factors such as plasmid copy number host strain inoculation antibiotic and type of culture medium Plasmid copy number Plasmids vary widely in their copy number per cell depending on
8. to avoid any injuries European Community risk and safety phrases are listed below for the components of the Invisorb Spin Plasmid Mini Two to which they apply are listed below as follows Solution C harmful H302 Harmful if swallowed H315 Causes skin irritation H319 Causes serious eye irritation P280 Wear protective gloves protective clothing eye protection face protection P305 P351 P338 IF IN EYES Rinse cautiously with water for several minutes Remove contact lenses if present and easy to do Continue rinsing Emergency medical information can be obtained 24 hours a day from infotrac outside of USA 1 352 323 3500 in USA 1 800 535 5053 5 Invisorb Spin Plasmid Mini Two 1015 Product characteristic of Invisorb Spin Plasmid Mini Two Starting material Time for preparation Ratio 0 5 2 0 ml bacteria suspension up to 20 ug lt 15 min A260 A 280 bacterial pellets from max 2 ml 1 8 2 1 suspension The Invisorb Spin Plasmid Mini Two allows for the purification of up to 20 ug plasmid DNA The kit provides a fast simple and cost effective plasmid mini preparation method for routine molecular biology laboratory application Harvested bacteria from an overnight culture are resuspended and processed by the alkaline lysis in the presence of RNAse followed by a neutralization step an adjustment of the binding conditions and after the clarification of the lysate from precipitated prote
9. ug ml Kanamycin 10 mg ml H2O 10 50 ug ml Streptomycin 10 mg ml H2O 10 50 ug ml Tetracycline 5 mg ml Ethanol 10 50 pg ml For further information see Molecular cloning a laboratory manual J Sambrook E F Fritsch T Maniatis Cold Spring Harbor NY Cold Spring Harbor Laboratory Press ISBN 0 87969 309 6 ISBN 0 87969 576 5 ISBN 0 87969 577 3 Edition 3 1 Chapter 1 and Appendix A 16 Invisorb Spin Plasmid Mini Two 1015 Ordering information Product Package Size Catalogue No Invisorb Spin Plasmid Mini Two 10 preparations 1010140900 Invisorb Spin Plasmid Mini Two 50 preparations 1010140200 Invisorb Spin Plasmid Mini Two 250 preparations 1010140300 Invisorb Spin Plasmid Mini Two 500 preparations 1010140400 Solution A 15 ml 1010146000 Solution B 15 ml 1010146100 Solution C 15 ml 1010146200 Wash Solution add 48 ml Ethanol 12 ml 1010143700 Elution Solution 15 ml 1010144000 Invisorb Plasmid HTS 96 Kit C 2 x 96 preparations 7010300200 Invisorb Plasmid HTS 96 Kit C 4 x 96 preparations 7010300300 Invisorb Plasmid HTS 96 Kit C 24 x 96 preparations 7010300400 InviMag Plasmid Kit KF96 1 x 96 preparations 7410300100 InviMag Plasmid Kit KF96 5 x 96 preparations 7410300200 17 Invisorb Spin Plasmid Mini Two 1015 stratecee molecular STRATEC Molecular GmbH Robert Rossle Str 10 13125 Berlin Germany Phone 49 30 94 89 29 01 Fax 49 30 94 89 29 09
10. 0 5 x 50 10 x 50 aa 10 50 5 x 50 10x 50 Manual 1 1 1 1 Initial steps Wash Solution Add 48 ml 96 100 Add 160 ml 96 100 Add 160 ml 96 100 is ready to use ethanol to the bottle Wash Solution ethanol to each bottle Wash Solution ethanol to each bottle Wash Solution Reagents and equipment to be supplied by user Microcentrifuge Measuring cylinder 250 ml Pipet and pipet tips Disposable gloves Reaction tubes 1 5 ml or 2 0 ml Vortexer 96 100 ethanol 0000000 Storage The Invisorb Spin Plasmid Mini Two should be stored dry at room temperature and is stable for at least 12 months under these conditions Make sure that all components are at room temperature If there are any precipitates in the reagents provided dissolve them by careful warming up to 37 C Room temperature RT is defined as ranging from 15 30 C 3 Invisorb Spin Plasmid Mini Two 1015 Symbols Manufacturer Lot number Catalogue number Expiry date mi ri KA EE 7 I Consult operating instructions 1 I Temperature limitation O Do not reuse Quality control and product warranty STRATEC Molecular warrants the correct function of the Invisorb Spin Plasmid Mini Two for applications as described in this manual Purchaser must determine the suitability of the Product for its particular use Should any Product fail to perform the applications as described in the manual STRATEC Molecul
11. 000 13 000 rpm Do not vortex 6 During centrifugation time place needed amount of Spin Filters into the 2 0 ml Receiver Tubes 7 Transfer decant the clarified supernatant into the Spin Filter Incubate for 1 min on to the Spin Filter Centrifuge for 1 min at 11 000 x g 11 000 rpm 8 Discard the filtrate Add 750 ul Wash Solution Centrifuge for 1 min at 11 000 x g 11 000 rpm Discard the filtrate 9 Centrifuge for 3 min at full speed 12 000 16 000 x g 11 000 13 000 rpm to complete removing the residual ethanol 10 Place the Spin Filter into a new 1 5 ml Receiver Tube and add 50 100 ul Elution Solution directly onto the center of the Spin Filter surface Incubate for 1 min at room temperature Finally centrifuge at 11 000 x g 11 000 rpm for 1 min to elute the plasmid DNA Note To increase the final DNA yield we recommend using a higher volume of Elution Solution A longer incubation time with Elution Buffer up to 10 min also leads to a slightly higher final yield In order to increase the DNA concentration we recommend eluting in a smaller volume than 50 Ll minimal 30 ul but this will lead to a reduction of yield Attention For in vitro transcription application please elute the plasmid DNA with daHzO Note The Elution Solution contains EDTA 12 Invisorb Spin Plasmid Mini Two 1015 Protocol 2 Purification of low copy plasmids amp cosmids from up to 10 ml bacteria cultures Please read the instr
12. NA 7 Removing residual contaminants 7 Elution of plasmid DNA 8 DNA Yield 8 Plasmid copy number 8 Host strains 8 Inoculation 8 Antibiotics 9 Culture media 9 Scheme 10 Important notes 11 Important points before starting a protocol 11 Preparing reagents and buffers 11 Growth of bacterial cultures 11 Instructions 12 Protocol 1 Preparation of pDNA from 0 5 2 0 ml bacteria cultures 12 Protocol 2 Purification of low copy plasmids amp cosmids from 10 ml bacteria culture 13 Protocol 3 Purification of plasmid DNA from 2 ml gram positive bacteria cultures 13 Troubleshooting 14 Appendix 15 General notes on handling pDNA 15 Examples of Plasmids Media Antibiotics and Bacterial trains 16 Common plasmid Vectors 16 Common media for bacterial culture 1L 16 Common antibiotics 16 Ordering information 17 2 Invisorb Spin Plasmid Mini Two 1015 Kit contents of Invisorb Spin Plasmid Mini Two Store all kit components at room temperature 10 pDNA 50 pDNA 250 pDNA 500 pDNA extractions extractions extractions extractions Catalogue No 1010140900 1010140200 1010140300 1010140400 Solution A 2x2ml 15 ml 70 ml 140 ml Solution B 2x2ml 15 ml 70 ml 140 ml Solution C 2x2ml 15 ml 70 ml 140 ml 10 ml 12 ml 1x40 ml 2x 40 ml Wash Solution ready to use final volume 60 ml final volume 1x 200 ml final volume 2x 200 ml Elution Solution 2ml 10 ml 30 ml 60 ml Spin Filter 10 50 5 x 50 10 x 50 Me 10 5
13. ar will check the lot and if STRATEC Molecular investigates a problem in the lot STRATEC Molecular will replace the Product free of charge STRATEC Molecular reserves the right to change alter or modify any Product to enhance its performance and design at any time In accordance with STRATEC Moleculars ISO 9001 2000 and ISO EN 13485 certified Quality Management System the performance of all components of the Invisorb Spin Plasmid Mini Two have been tested separately against predetermined specifications routinely on lot to lot to ensure consistent product quality If you have any questions or problems regarding any aspects of Invisorb Spin Plasmid Mini Two or other STRATEC Molecular products please do not hesitate to contact us A copy of STRATEC Molecular s terms and conditions can be obtained upon request or are presented at the STRATEC Molecular webpage For technical support or further information please contact from Germany 49 0 30 9489 2901 2910 from abroad 49 0 30 9489 2907 or contact your local distributor Intended use The Invisorb Spin Plasmid Mini Two provides a fast efficient robust and low cost purification system for plasmid DNA from 0 5 ml to 2 ml of overnight bacterial cultures or bacterial pellets The Invisorb Spin Plasmid Mini Two combines the alkaline lysis procedure for the isolation of plasmid DNA with the very efficient binding of plasmid DNA onto a Spin Filter surface The isolation protocol as well as
14. from a freshly streaked selective plate Subculturing directly from glycerol stocks agar stabs and liquid cultures may lead to uneven plasmid yield or loss of the plasmid Inoculation from plates that have been stored for a long time may also lead to loss or mutation of the plasmid The desired clone should be streaked from a glycerol stock onto a freshly prepared agar plate containing the appropriate selective agent so that single colonies can be isolated This procedure should then be repeated to ensure that a single colony of an antibiotic resistant clone can be picked A single colony should be inoculated into 1 5 ml of media containing the appropriate selective agent and grown with vigorous shaking for 12 16 hours Growth for more than 16 hours is not recommended since bacterial culture overgrows and plasmid yields may be reduced 8 Invisorb Spin Plasmid Mini Two 1015 Antibiotics Antibiotic selection should be applied at all stages of growth Many plasmids in use today do not contain the par locus which ensures that the plasmids segregate equally during cell division Daughter cells that do not receive plasmids will replicate much faster than plasmid containing cells in the absence of selective pressure and can quickly take over the culture The stability of the selective agent should also be taken into account Resistance to ampicillin for example is mediated by B lactamase which is encoded by the Plasmid linked bla gene and which hydr
15. fugation steps were made with the Centrifuge 5415 D from Eppendorf The indicated rpm amounts refer to this centrifuge Protocol 1 Preparation of PDNA from 0 5 2 0 ml bacteria cultures Please read the instructions carefully and conduct the prepared procedure 1 Transfer from 0 5 ml up to 2 ml of the overnight E coli culture into a 1 5 ml or 2 0 ml microcentrifuge tube For larger volumes of starting material please contact our technical support 2 Centrifuge for 1 min at maximum speed 12 000 16 000 g 11 000 13 000 rpm to pellet the cells remove the supernatant as completely as possible 3 Resuspend the cell pellet in 250 ul Solution A completely by vortexing or by pipetting up and down For multiple processing of samples we recommend a vortexer Note No cell pellet or clumps should be visible 4 Add 250 ul Solution B close the tube and mix carefully by inverting the tube 5 times Do not perform the lysis step more than 5 min Important Do not vortex the tube to mix the suspension This step is critical for the separation of bacterial chromosomal DNA from plasmid DNA Mechanical stress by vortexing or extensive mixing leads to shearing of high molecular weight chromosomal DNA This sheared chromosomal DNA is not precipitated by NaOH SDS and contaminates the plasmid DNA 5 Add 250 ul Solution C and mix gently but thoroughly by shaking the tube 4 6 times Centrifuge for 5 min at full soeed 12 000 16 000 g 11
16. ins and cell unlysed debris the pDNA binds to the membrane of the spin filter All impurities are efficiently removed by washing and the ultra pure pDNA is eluted in a low salt buffer The kit provides reproducible recovery rates of highly purified pDNA The hands on time necessary for the whole procedure is reduced to a minimum Due to the high purity the isolated pDNA is ready to use in downstream applications such as PCR Enzyme Digestion Seguencing Cloning In vitro translation OOO 0 0 To purify plasmid DNA in convenient 96 well format STRATEC Molecular offers the Invisorb Plasmid HTS 96 Kit for use in centrifuge The PCR method is covered by U S Patents 4 683 195 and 4 683 202 owned by Hoffmann LaRoche Inc The purchase of the Invisorb Spin Plasmid Mini Two cannot be construed as an authorization or implicit licence to practice PCR under any patents held by Hoffmann LaRoche Inc 6 Invisorb Spin Plasmid Mini Two 1015 Principle and procedure The Invisorb Spin Plasmid Mini Two procedure is comprised of following steps growth of bacterial culture alkaline lysis of cells precipitation of proteins and genomic DNA adsorption of plasmid DNA to the membrane of the spin column removal of contaminants and elimination of ethanol elution of plasmid DNA ARVNA All steps are performed without use of phenol chloroform CsCI ethidium bromide and without alcohol precipitation This manual contains three prot
17. lecular does not provide for validation of performance characteristics of the Product with respect to specific applications STRATEC Molecular Products may be used e g in clinical diagnostic laboratory systems conditioned upon the complete diagnostic system of the laboratory the laboratory has been validated pursuant to CLIA 88 regulations in the U S or equivalents in other countries All Products sold by STRATEC Molecular are subject to extensive quality control procedures according to ISO 9001 2000 and ISO EN 13485 and are warranted to perform as described herein Any problems incidents or defects shall be reported to STRATEC Molecular immediately upon detection thereof The chemicals and the plastic parts are for laboratory use only they must be stored in the laboratory and must not be used for purposes other than intended The Product with its contents is unfit for consumption Safety information when and while working with chemicals always wear a suitable lab coat disposable gloves and protective goggles Avoid skin contact Adhere to the legal requirements for working with biological material For more information please consult the appropriate material safety data sheets MSDS These are available online in convenient and compact PDF format at www stratec com for each STRATEC Molecular Product and its components If buffer bottles are damaged or leaking WEAR GLOVES AND PROTECTIVE GOGGLES when discarding the bottles in order
18. ocols Lysis After harvesting and resuspending using Solution A the bacterial cells are lysed in the presence of Solution B Solution A contains RNase which digests the RNA during lysis SDS solubilizes the proteins and phospholipids of the cell membrane This leads to the lysis of the cells and the release of cell contents During the subsequent neutralization of the lysate chromosomal and pDNA as well as proteins will be denatured The optimal lysis time allows maximum release of the pDNA without release of chromosomal DNA Attention Long exposure of the pDNA to alkaline conditions may cause the plasmid to become irreversibiy denaturated This denaturated form of pDNA runs faster on agarose gel and is resistant to restriction enzyme digestion During this step the lysate is neutralized and adjusted to binding conditions in one step by the addition of Solution C The proteins are denatured chromosomal DNA cellular debris and SDS are precipitated while the smaller plasmid DNA renatures correctly and stays in the solution It is important that the solution is mixed thoroughly and gently to ensure complete precipitation Attention To prevent contamination of plasmid DNA with chromosomal DNA vigorous shaking and vortexing must be avoided during lysis Separation of plasmid from chromosomal DNA is based on coprecipitation of the cell wall bound chromosomal DNA with insoluble complexes containing salt detergent and protein Plasmid DNA remains
19. olyzes ampicillin Levels of ampicillin in the culture medium are thus continually depleted This phenomenon is clearly demonstrated on ampicillin plates where satellite colonies appear as the ampicillin is hydrolyzed in the vicinity of a growing colony Ampicillin is also very sensitive to temperature in solution it should be stored frozen in single use aliquots Culture media Luria Bertani LB broth is the recommended culture medium for use with Invisorb Spin Plasmid Kits since richer broths such as TB Terrific Broth or 2x YT lead to extremely high cell densities which can overload the purification system It should be noted that cultures grown in TB may yield 2 5 times the number of cells compared to cultures grown in LB broth If these media are used recommended culture volumes must be reduced to match the capacity of the Spin Filter membrane If excess culture volume is used alkaline lysis will be inefficient the Spin Filter membrane will be overloaded and the performance of the system will be unsatisfactory Furthermore the excessive viscosity of the lysate will require vigorous mixing which may result in shearing of bacterial genomic DNA and contamination of the plasmid DNA Care must also be taken if strains are used which grow unusually fast or to very high cell densities In such cases doubling the volumes of Solution A B and C may be beneficial It is best to calculate culture cell density and adjust the volume accordingly
20. r 10 min at 37 C Follow protocol 1 starting at step 4 13 Invisorb Spin Plasmid Mini Two 1015 Troubleshooting Problem Cause Comments and suggestions low yield of plasmid DNA incorrect Wash Solution poor elution of pDNA conditions for bacterial cultures are not optimal elution Solution incorrect too much starting material prepare the Wash Solution exactly as described in the manual don t forget the ethanol Storage of Wash Solution with firmly fixed cap add the Elution Solution directly on the center of the Spin Filter changing of the conditions media growing time and other DNA is eluted only using low salt buffer e g Elution Solution or water Elution efficiency depends on pH maximum efficiency is achieved between pH 7 0 8 0 When using water for elution make sure that the pH is within this range use only the recommended amount of bacterial suspension contamination of the plasmid DNA with chromosomal DNA problems because of poor cleavage by restriction endonucleases or for other applications the sample was mixed too vigorously during step 4 or step 5 bacteria overgrown lysis too long contamination of the final plasmid DNA with salt components contamination of the final DNA with ethanol follow exactly the protocol Do not vortex at these steps Mix the samples only by inverting the tubes carefully reduce time for growing the bacterial culture lysi
21. s must not exceed 5 minutes wash the plasmid DNA bound on the Spin Filters as described keep the given centrifugation time step 9 extend it if necessary test the smell RNA contamination RNAse digestion insufficient check culture volume against recommended volumes and reduce if necessary add more RNase A recover DNA by precipitating the eluate digesting with RNase A and purifying on a new spin column additional band below the super coiled plasmid DNA band denaturated super coiled plasmid DNA 14 incorrect incubation in Solution B increased incubation time with Solution B can cause denaturation of super coiled plasmid DNA Invisorb Spin Plasmid Mini Two 1015 Appendix General notes on handling PDNA Nature of DNA The length and delicate physical nature of DNA require careful handling to avoid damage due to shearing and enzymatic degradation Other conditions that affect the integrity and stability of DNA include acidic and alkaline environments high temperature and UV irradiation Careful isolation and handling of DNA is necessary to ensure it will function well in various downstream applications Damaged DNA could perform poorly in applications such as Southern blotting and long template PCR Handling fresh and stored material before the extraction of pDNA For the isolation of plasmid DNA from bacteria use either fresh cultures or pellets that have been frozen and stored at 20 C
22. spin filters into labeled 2 0 ml receiver tubes 3 Label the needed amount of 1 5 ml receiver tubes per sample 1 receiver tube 10 DNA extractions Wash Solution is ready to use 50 DNA extractions Add 48 ml 96 100 ethanol to the bottle Wash Solution 250 DNA extractions Add 160 ml 96 100 ethanol to the bottle Wash Solution 500 DNA extractions Add 160 ml 96 100 ethanol to each bottle Wash Solution Important note Incomplete removal of the bacteria culture medium will affect lysis and dilute the lysate Growth of bacterial cultures Pick a single colony from a freshly streaked selective plate and inoculate a culture of 1 5 ml LB medium containing the appropriate selective antibiotic Incubate for 12 16 h at 37 C with vigorous shaking Growth for more than 16 h is not recommended since cells begin to lyse and plasmid yields may be reduced Use a tube or flask with a volume of at least 4 times the volume of the culture Harvest the bacterial cells by centrifugation at minimum 6 000 x g 8 000 rpm in a table top microcentrifuge for 3 min at room temperature The bacterial cells can also be harvested in 15 ml centrifuge tubes at 5 400 x g 7 000 rpm for 10 min at 4 C Remove all traces of supernatant by inverting the open centrifuge tube until all medium has been drained 11 Invisorb Spin Plasmid Mini Two 1015 Instructions The following notes are valid for all protocols Note The centri
23. stratecee molecular 7 User manual Invisorb Spin Plasmid Mini Two for isolation of pDNA from 0 5 2 0 ml bacteria suspension 1010140X00 pal STRATEC Molecular GmbH D 13125 Berlin Instruction for Invisorb Spin Plasmid Mini Two The Invisorb Spin Plasmid Mini Two provides an extremely fast and very convenient tool for an efficient purification of low and high copy plasmid DNA from 0 5 to 2 ml bacteria suspension in comfortable spin format Trademarks Invisorb InviMag Registered marks trademarks etc used in this document even when not specifically marked as such are not to be considered unprotected by law The Invisorb technology is covered by patents and patent applications US 6 110363 US 6 043 354 US 6 037 465 EP 0880535 WO 9728171 WO 9534569 EP 0765335 DE 19506887 DE 10041825 2 WO 0034463 Invisorb and InviMag are registered trademarks of STRATEC Biomedical AG The PCR process is covered by US Patents 4 683 195 and 4 683 202 and foreign equivalents owned by Hoffmann La Roche AG 2015 STRATEC Molecular all rights reserved 1 Invisorb Spin Plasmid Mini Two 1015 Contents Kit contents of Invisorb Spin Plasmid Mini Two 3 Reagents and equipmet to be supplied by user 3 Storage 3 Symbols 4 Quality control 4 Intended use 4 Product use limitation 5 Safety information 5 Product characteristic of Invisorb Spin Plasmid Mini Two 6 Principle and procedure 7 Lysis 7 Binding plasmid D
24. their origin of replication e g pMB1 ColE1 or pSC101 which determines whether they are under relaxed or stringent control and depending on the size of the plasmid and its associated insert Some plasmids such as the pUC series and derivatives have mutations which allow them to reach very high copy numbers within the bacterial cell Plasmids based on pBR322 and cosmids are generally present in lower copy numbers Very large plasmids and cosmids are often maintained at very low copy numbers per cell Host strains Most E coli strains can be used successfully to isolate plasmid DNA although the strain used to propagate a plasmid has an effect on the quality of the purified DNA Host strains such as DH DH5a and C600 give high quality DNA The slower growing strain XL1 Blue also yields DNA of very high quality which works extremely well for sequencing Strain HB101 and its derivatives such as TG1 and the JM series produce large amounts of carbohydrates which are released during lysis and can inhibit enzyme activities if not completely removed In addition these strains have high levels of endonuclease activity which can reduce DNA quality The methylation and growth characteristics of the strain should also be taken into account when selecting a host strain XL1 Blue and DH5a are highly recommended for reproducible and reliable results Inoculation Bacterial cultures for plasmid preparation should always be grown from a single colony picked
25. uctions carefully and conduct the prepared procedure 1 Transfer 1 10 ml of the overnight E coli cultures grown in LB medium into a 15 ml Falcon Tube and spin down the bacterial cells Follow the protocol 1 but Note When using 10 mi culture volume it is recommended to double the volumes of Solution A B C When plasmids or cosmids are bigger than 10 kb prewarm Elution Solution or water to 70 C prior eluting DNA from the Spin Filter membrane A 10 ml overnight LB culture typically yields 5 10 ug DNA Attention this is an additional application and the needed buffer volumes are not calculated in the provided buffers Please order additional buffer if you would like to perform this application in larger guantities see Ordering information page 19 Protocol 3 Purification of plasmid DNA from 2 ml gram positive bacteria cultures Please read the instructions carefully and conduct the prepared procedure 1 Transfer 0 5 ml up to 2 ml of the overnight culture into a 1 5 ml or 2 0 ml microcentrifuge tube 2 Centrifuge for 1 min at maximum speed 12 000 16 000 g 11 000 13 000 rpm to pellet the cells remove the supernatant as completely as possible 3 Resuspend the cell pellet in 250 pl Solution A completely by vortexing or by pipetting up and down Add 10 ul of lysozyme 10mg ml or according the producers instructions Mix the suspension For multiple processing of samples we recommend a vortexer 4 Incubate fo
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