EZNA® Plasmid DNA Mini Kit I EZNA® Plasmid - Omega Bio-Tek
Contents
1. Letsit at room temperature for 1 minute 26 Centrifuge at maximum speed for 1 minute Note This represents approximately 7096 of bound DNA An optional second elution will yield any residual DNA though at a lower concentration 27 Store DNA at 20 C E Z N A Plasmid DNA Mini Kit II Spin Protocol E Z N A Plasmid Mini Kit II Protocol Spin Method All centrifugation should be performed at room temperature unless otherwise noted For low copy number plasmids refer to Page 21 This protocol is designed to isolate plasmid DNA from E coli grown in 10 15 mL LB culture Materials and Equipment to be Supplied by User 10096 ethanol Microcentrifuge capable of at least 13 000 x g Nuclease free 2 mL microcentrifuge tubes Centrifuge capable of at least 5 000 x g with swing buckets Appropriate centrifuge tube for Step 1 e Optional sterile deionized water e Optional water bath or incubator capable of 70 C e Optional 3M NaOH solution Before Starting e Heat Elution Buffer to 70 C if plasmid DNA is gt 10kb Prepare DNA Wash Buffer and Solution 1 according to the instructions in the Preparing Reagents section on Page 6 1 Isolate a single colony from a freshly streaked selective plate and inoculate a culture of 10 15 mL 50 ug mL LB medium containing the appropriate selective antibiotic Incubate for 12 16 hours at 37 C with vigorous shaking 300 rpm Use culture tube or a flask with a volume of a2. Page 24 for ordering information 1 Increase the volume of starting culture from that of high copy number plasmids Use 5 10 mL bacterial culture for the E Z N A Plasmid DNA Mini Kit or 20 30 mL bacterial culture for E Z N A Plasmid DNA Mini Kit Il 2 Pellet the bacterial cells by centrifugation 3 Decant or aspirate and discard the culture media 4 Perform Steps 4 8 in the standard protocols with double the volumes of Solution I Solution ll and Solution III 5 Continue with Step 9 of the standard protocols by following the wash drying and elution steps There is no need to increase the volumes of HB Buffer DNA Wash Buffer or Elution Buffer 21 Troubleshooting Guide Please use this guide to troubleshoot any problems that may arise For further assistance please contact the technical support staff toll free at 800 832 8896 Possible Problems and Suggestions Low DNA yields Only use LB or YT medium containing ampicillin Do not use more than 5 mL high copy number plasmids or 10 mL low copy number plasmids culture with the basic protocols Cells may not have been dispersed adequately prior to the addition of Solution Il Vortex to completely resuspend the cells Increase Solution Il incubation time to obtain a clear lysate Solution Il if not tightly closed may need to be replaced Poor cell lysis Do not incubate cultures for more than 16 hours at 37 C Storage of cultures for extended periods
3. containing impurities Plasmid DNA is contaminated with RNA RNase A treatment is insufficient Background reading is high due to silica fine particulates Purification is incomplete due to column overloading Check the absorbance of the ethanol between 250 nm and 300 nm Do not use ethanol with high absorbance Traces of impurities may remain on the binding column after washing and contribute to the absorbance in the final product Confirm that the RNase A Solution was added to Solution I prior to first use The RNase A solution may degrade due to high temperatures gt 65 C or prolonged storage gt 6 months at room temperature Spin the DNA sample at maximum speed for 1 minute use the supernatant to repeat the absorbance readings Reduce the initial volume of culture 23 Ordering Information The following components are available for purchase separately Call Toll Free at 1 800 832 8896 Product Part Number DNase RNase free microcentrifuge tubes 1 5 mL 500 pk 10 pk cs SSI 1210 00 DNase RNase free microcentrifuge tubes 2 0 mL 500 pk 10 pk cs SSI 1310 00 Vacuum Manifold VAC 08 HiBind DNA Mini Columns 200 DNACOL 02 Solution 250 mL PS001 Solution Il 250 mL PS002 Solution III 250 mL PS003 Elution Buffer 100 mL PDRO48 HB Buffer 250 mL PS009 DNA Wash Buffer 100 mL PS010 RNase A 400 uL AC117 HiBind E Z N A and MicroElute are registered trademarks of Omega Bio tek Inc Q
4. ethanol may interfere with downstream applications Transfer the HiBind DNA Mini Column to a clean 1 5 mL microcentrifuge tube Add 80 100 uL Elution Buffer or sterile deionized water directly to the center of the column membrane Note The efficiency of eluting DNA from the HiBind DNA Mini Column is dependent on pH If using sterile deionized water make sure that the pH is around 8 5 19 23 24 25 20 E Z N A Plasmid DNA Mini Kit II Spin Protocol Let sit at room temperature for 1 minute Centrifuge at maximum speed for 1 minute Note This represents approximately 7096 of bound DNA An optional second elution will yield any residual DNA though at a lower concentration Store DNA at 20 C Low Copy Number Plasmid and BAC DNA Protocol E Z N A Plasmid Mini Kit Protocol Low Copy Number Plasmid and BAC DNA Protocol Low copy number plasmids generally give 0 1 1 ug DNA per mL overnight culture For the isolation of plasmid DNA from low copy number plasmids 0 1 1 ug mL culture or low copy number plasmid 1 2 ug mL culture bacteria use the following modified protocol Note The E Z N A Plasmid DNA Mini Kit and the E Z N A Plasmid DNA Mini Kit II come with enough Solution I Solution Il and Solution III to perform the standard protocols Additional Solution I Solution Il and Solution Ill are needed to perform the Low Copy Number Plasmid and BAC DNA Protocol These buffers can be purchased separately See
5. 1 D6942 02 D6943 02 100 mL D6945 02 3 Check Solution Il and Solution Ill for precipitation before use Redissolve any precipitation by warming to 37 C Storage and Stability All of the E Z N A Plasmid DNA Mini Kit and Plasmid DNA Mini Kit II components are guaranteed for at least 12 months from the date of purchase when stored as follows Solution once RNase A is added should be stored at 2 8 C All other materials should be stored at room temperature Solution Il must be tightly capped when not in use Guidelines for Vacuum Manifold The following is required for use with the Vacuum Spin Protocol A Vacuum Manifold We recommend Omega Bio tek s VAC 08 Other Compatible Vacuum Manifolds Qiagen OlAvac24 Sigma AldrichVM20 Promega Vacman or manifold with standard Luer connector B Vacuum Flask C Vacuum Tubing D Vacuum Source review tables below for pressure settings Manifold Recommended Pressure mbar VAC 08 200 to 600 Conversion from millibars Multiply by Millimeters of mercury mmHg 0 75 Inches of mercury inch Hg Tors or Atmospheres atmos Pounds per Square Inch psi Vacuum Setup dH s Hss Omega Bio tek s VAC 08 C Vacuum Tubing D Vacuum Source A Vacuum Manifold B Vacuum Flask Recommended Settings Growth and Culture of Bacteria Bacterial Strain Selection It is strongly recommended that an end A negative strain of E coli be used for routine p
6. NA Mini Kits are specially designed for use with cultures grown in Luria Bertani LB medium Richer broths such as TB Terrific Broth or 2xYT lead to high cell densities that can overload the purification system and therefore are not recommended If rich media has to be used growth times have to be optimized and the recommended culture volumes must be reduced to match the capacity of the HiBind DNA Mini Column Note As culture ages DNA yield may begin to decrease due to cell death and lysis within the culture Recommended Settings Culture Volume and Cell Density Do Not Exceed Maximum Recommended Culture Volumes For optimal plasmid yields the starting culture volume should be based on culture cell density A bacterial density between 2 0 and 3 0 at OD is recommended When using nutrient rich media care should be taken to ensure that the cell density does not exceed an OD of 3 0 Using a high density culture outside of the recommended OD range may overload the purification system E Z N A Plasmid DNA Mini Kit I Spin Protocol E Z N A Plasmid DNA Mini Kit Protocol Spin Protocol All centrifugation should be performed at room temperature unless otherwise noted For low copy number plasmids refer to Page 21 This protocol is designed to isolate plasmid DNA from E coli grown in an overnight 1 5 mL LB culture Materials and Equipment to be Supplied by User 10096 ethanol Microcentrifuge capable of at least 13 000 x g Nuclea
7. edium and binding capacity of the kit Of these factors the vector copy number culture volume and kit binding capacity are most important Plasmid copy number ranges from one copy to several hundred copies per cell as dictated by their origin of replication Very large plasmids often display a very low copy number per cell The expected yield of 5 mL overnight cultures LB medium with the E Z N A Plasmid Mini Kit are indicated in the following table Sample yields from a 5 mL starting culture Plasmid Rein Copy Number Expected eld pSC101 and its derivatives pGEM pMB1 300 700 10 20 ug Spin Protocol CN RI Pellet by Centrifugation Resuspend and Lyse Neutralize Clear Lysate Transfer Lysate to HiBind DNA Mini Column Bind Wash 3X Dry Elute Vacuum Spin Protocol Pellet by Centrifugation Resuspend and Lyse y g Neutralize Q Clear Lysate Transfer Lysate to HiBind DNA Mini Column J g and i Wash 3X pap CIO gt pee CO wea e Dry Elute Kit Contents D6943 00 D6943 01 D6943 02 z 2 2 2 7 50 00 50 00 50 00 120 mL Preparing Reagents 1 Add the vial of RNase A to the bottle of Solution and store at 2 8 C 50 and 200 prep size only 2 Dilute DNA Wash Buffer with 10096 ethanol as follows and store at room temperature 10096 Ethanol to be Added D6942 00 D6943 00 6mL D6945 00 D6942 01 D6943 01 60 mL D6945 0
8. f OMEGA Innovations in nucleic acid isolation Product Manual E Z N A Plasmid DNA Mini Kit D6942 00 5 preps Q Spin D6942 01 50 preps Q Spin D6942 02 200 preps Q Spin D6943 00 5 preps V Spin D6943 01 50 preps V Spin D6943 02 200 preps V Spin E Z N A Plasmid DNA Mini Kit II D6945 00 5 preps V Spin D6945 01 50 preps V Spin D6945 02 200 preps V Spin March 2013 For research use only Not intended for diagnostic testing E Z N A Plasmid DNA Mini Kit I E Z N A Plasmid DNA Mini Kit I Table of Contents WRIA eO CON aedis inso civit dotem dieron n edic Du reset ipa tei de 2 Yield ard Quality OF DNAst 3 Illustrated PIOFIOCOLS eu eU Deci niasa 4 PIE GO al MES rate esas tL co ec E 5 Preparing Reagents Storage and Stability s 6 Guidelines Tor Vacuum Manlfolg ssecestrre trii terere vto 7 Recommended Settings dcin madri sd qt Pocta Pn und 8 Plasmid DNA Mini Kit I Centrifugation Protocol 10 Plasmid DNA Mini Kit I Vacuum Protocol sss 13 Plasmid DNA Mini Kit II Centrifugation Protocol 17 Low Copy Number Plasmid Protocol eae 21 Troubleshooting GUB cma ir S RIPE EU RIOT EON SUPR 22 yea NUN T 24 Manual Revision March 2013 5 OMEGA bio tek Innovations in nucleic acid isolation Introduction The E Z N A family of products is an innovative system that radically simplifies the extrac
9. iBind DNA Mini Column Be careful not to disturb the pellet and that no cellular debris is transferred to the HiBind DNA Mini Column Turn on the vacuum source to draw the sample through the column Turn off the vacuum 14 15 16 17 18 19 20 21 22 23 24 E Z N A Plasmid DNA Mini Kit Vacuum Protocol Add 500 uL HB Buffer Turn on the vacuum source to draw the buffer through the column Turn off the vacuum Add 700 uL DNA Wash Buffer Note DNA Wash Buffer must be diluted with 10096 ethanol prior to use Please see Page 6 for instructions Turn on the vacuum source to draw the buffer through the column Turn off the vacuum Repeat Steps 17 19 for a second DNA Wash Buffer wash step Transfer the HiBind DNA Mini Column to a 2 mL Collection Tube Centrifuge the empty HiBind DNA Mini Column for 2 minutes at maximum speed to dry the column matrix Note It is important to dry the HiBind DNA Mini Column matrix before elution Residual ethanol may interfere with downstream applications Transfer the HiBind DNA Mini Column to a clean 1 5 mL microcentrifuge tube Add 30 100 pL Elution Buffer or sterile deionized water directly to the center of the column membrane Note The efficiency of eluting DNA from the HiBind DNA Mini Column is dependent on pH If using sterile deionized water make sure that the pH is around 8 5 15 16 E Z N A Plasmid DNA Mini Kit Vacuum Protocol 25
10. iagen OlAvac and Vacman are all trademarks of their respective companies PCR is a patented process of Hoffman La Roche Use of the PCR process requires a license 24
11. ience Equilibration Buffer is replaced with 3M NaOH provided by the user Q spin column vs V spin column The E Z N A Plasmid DNA Mini Kit is available with two different types of columns V spin columns have an attached cap while Q spin columns are capless The columns are otherwise identical in use and application Either column can be used with either the vacuum or centrifugation protocol Protocols The E Z N A Plasmid DNA Mini Kits are designed for fast and efficient processing Depending on the protocol the E Z N A Plasmid Mini Kits can be used with any microcentrifuge or vacuum manifold with standard luer connectors Yield and Quality of DNA Determine the absorbance of an appropriate dilution 20 to 50 fold of the sample at 260 nm and then at 280 nm The DNA concentration is calculated as follows DNA concentration Absorbance 260 x 50 x Dilution Factor pg mL A ratio greater than 1 8 indicates greater than 90 nucleic acid Alternatively quantity as well as quality sometimes can be determined best by agarose gel ethidium bromide electrophoresis by comparison to DNA samples of known concentrations Typically the majority of the DNA eluted is in monomeric supercoil form though concatemers may also be present Plasmid Copy Number and Expected Yield Yield and quality of the plasmid DNA obtained depends on a number of factors including plasmid copy number size of insert host strain culture volume culture m
12. lasmid isolation Examples of such strains include DH5a DH1 and C600 These host strains yield high quality DNA with E Z N A Plasmid DNA Mini Kit Protocols XL1 Blue although a slower growing strain is also recommended due to its yield of high quality DNA Host strains derivatives from HB101 such as TG1 and the JM100 series release large amounts of carbohydrates during lysis which may inhibit enzyme activity when not completely removed Some strains may also lower DNA quality due to having high levels of endonuclease activity and therefore are not recommended i e JM101 JM110 HB101 One may reduce the amount of culture volume or double the volumes of Solution I Solution II and Solution III if problems are encountered with strains such as TG1 and Top10F Inoculation Bacterial cultures for plasmid preparations should always be grown from a single colony picked from a freshly streaked plate Subculturing directly from glycerol stock or liquid cultures may lead to uneven yields or plasmid loss Optimal results are obtained by using one single isolated colony from a freshly transformed or freshly streaked plate to inoculate an appropriate volume of starter culture containing the appropriate antibiotic and then incubated for 12 16 at 37 C with vigorous shaking 300 rpm shaking incubator Note Aeration is very important The culture volume should not exceed 1 4 the volume of the container Culture Media The E Z N A Plasmid D
13. o isolate plasmid DNA from E coli grown in an overnight 1 5 mL LB culture See Page 7 for guidelines on preparing the vacuum manifold used in this protocol Materials and Equipment to be Supplied by User Vacuum Manifold 10096 ethanol Microcentrifuge capable of at least 13 000 x g Nudlease free 1 5 mL or 2 mL microcentrifuge tubes Appropriate centrifuge and centrifuge tube for Step 1 e Optional sterile deionized water e Optional water bath or incubator capable of 70 C e Optional 3M NaOH solution Before Starting Heat Elution Buffer to 70 C if plasmid DNA is gt 10 kb Prepare DNA Wash Buffer and Solution according to the instructions in the Preparing Reagents section on Page 6 1 Isolate a single colony from a freshly streaked selective plate and inoculate a culture of 1 5 mLLB medium containing the appropriate selective antibiotic Incubate for 12 16 hr at 37 C with vigorous shaking 300 rpm Use a 10 20 mL culture tube or a flask with a volume of at least 4 times the volume of the culture It is strongly recommended that an endA negative strain of E coli be used for routine plasmid isolation Examples of such strains include DH5a and JM109 2 Centrifuge at 10 000 x g for 1 minute at room temperature 3 Decant or aspirate and discard the culture media 4 Add 250 uL Solution I RNase A Vortex or pipet up and down to mix thoroughly Complete resuspension of cell pellet is vital for
14. obtaining good yields Note RNase A must be added to Solution before use Please see the instructions in the Preparing Reagents section on Page 6 13 E Z N A Plasmid DNA Mini Kit Vacuum Protocol Transfer suspension into a new 1 5 mL microcentrifuge tube Add 250 uL Solution Il Invert and gently rotate the tube several times to obtain a clear lysate A 2 3 minute incubation may be necessary Note Avoid vigorous mixing as this will shear chromosomal DNA and lower plasmid purity Do not allow the lysis reaction to proceed more than 5 minutes Store Solution Il tightly capped when not in use to avoid acidification from CO in the air Add 350 uL Solution Ill Immediately invert several times until a flocculent white precipitate forms Note It is vital that the solution is mixed thoroughly and immediately after the addition of Solution III to avoid localized precipitation Centrifuge at maximum speed 213 000 x g for 10 minutes A compact white pellet will form Promptly proceed to the next step Prepare the vacuum manifold according to manufacturer s instructions 10 Connect the HiBind DNA Mini Column to the vacuum manifold Optional Protocol for Column Equilibration 11 12 13 14 1 Add 100 uL 3M NaOH to the HiBind DNA Mini Column 2 Turnon the vacuum source to draw the NaOH through the column 3 Turn off the vacuum Transfer the cleared supernatant from Step 8 by CAREFULLY aspirating it into the H
15. oceed to the next step Insert a HiBind DNA Mini Column into a 2 mL Collection Tube Optional Protocol for Column Equilibration 10 11 12 18 1 Add 100 uL 3M NaOH to the HiBind DNA Mini Column 2 Centrifuge at maximum speed for 30 60 seconds 3 Discard the filtrate and reuse the collection tube Transfer 700 uL cleared lysate from Step 8 by CAREFULLY aspirating it into the HiBind DNA Mini Column Be careful not to disturb the pellet and that no cellular debris is transferred to the HiBind DNA Mini Column Centrifuge at maximum speed for 1 minute Discard the filtrate and reuse the collection tube 13 14 15 16 17 18 19 E Z N A Plasmid DNA Mini Kit II Spin Protocol Repeat Steps 10 12 until all cleared lysate has been transferred to the HiBind DNA Mini Column Add 500 uL HB Buffer Centrifuge at maximum speed for 1 minute Discard the filtrate and reuse collection tube Add 700 uL DNA Wash Buffer Note DNA Wash Buffer must be diluted with 10096 ethanol prior to use Please see Page 6 for instructions Centrifuge at maximum speed for 1 minute Discard the filtrate and reuse the collection tube Optional Repeat Steps 17 19 for a second DNA Wash Buffer wash step 20 21 22 Centrifuge the empty HiBind DNA Mini Column for 2 minutes at maximum speed to dry the column matrix Note It is important to dry the HiBind DNA Mini Column matrix before elution Residual
16. prior to plasmid isolation is detrimental Low elution efficiency The pH of Elution Buffer or water must be pH 8 0 9 0 Culture is overgrown or not fresh Such plasmids may yield as little as 0 1 ug DNA from a 1 mL overnight culture Double the culture volume and follow the low copy number plasmid protocol on Page 21 Low copy number plasmid used Follow the Optional Protocol for Column Equilibration prior Column matrix lost to transferring the cleared lysate to the HiBind DNA Mini binding capacity Column Add 100 uL 3M NaOH to the column prior to loading during storage the sample Centrifuge at 10 000 x g for 30 seconds Discard the filtrate No DNA eluted DNA Wash Buffer not diluted with ethanol High molecular weight DNA contamination of product Over mixing of cell lysate upon addition Do not vortex or mix aggressively after adding Solution Il of Solution II Prepare DNA Wash Buffer according to instructions on Page 6 22 Culture overgrown Troubleshooting Guide Overgrown cultures contain lysed cells and degraded DNA Do not grow cell longer than 16 hours Plasmid DNA floats out of well while loading agarose gel Ethanol was not completely removed from column following wash steps Centrifuge column as instructed to dry the column before elution Absorbance of purified DNA does not accurately reflect quantity of the plasmid A A ratio is high or low DNA Wash Buffer is diluted with ethanol
17. se free 1 5 mL or 2 mL microcentrifuge tubes Culture tubes Optional sterile deionized water Optional water bath or incubator capable of 70 C Optional 3M NaOH solution Before Starting 10 Heat Elution Buffer to 70 C if plasmid DNA is gt 10 kb Prepare DNA Wash Buffer and Solution according to the instructions in the Preparing Reagents section on Page 6 Isolate 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 hours at 37 C with vigorous shaking 300 rpm Use a 10 20 mL culture tube or a flask with a volume of at least 4 times the volume of the culture It is strongly recommended that an endA negative strain of E coli be used for routine plasmid isolation Examples of such strains include DH5a and JM109 Centrifuge at 10 000 x g for 1 minute at room temperature Decant or aspirate and discard the culture media Add 250 uL Solution I RNase A Vortex or pipet up and down to mix thoroughly Complete resuspension of cell pellet is vital for obtaining good yields Note RNase A must be added to Solution before use Please see the instructions in the Preparing Reagents section on Page 6 E Z N A Plasmid DNA Mini Kit Spin Protocol Transfer suspension into a new 1 5 mL microcentrifuge tube Add 250 uL Solution Il Invert and gently rotate the tube several times to obtain a clear lysate A 2 3 minu
18. t least 4 times the volume of the culture It is strongly recommended that an end A negative strain of E coli be used for routine plasmid isolation Examples of such strains include DH5a and JM109 2 Centrifugation at 5 000 x g for 10 minutes at room temperature 3 Decant or aspirate the medium and discard 4 Add 500 uL Solution I RNase A Vortex or pipet up and down to mix thoroughly Complete resuspension of cell pellet is vital for obtaining good yields Note RNase A must be added to Solution before use Please see the instructions in the Preparing Reagents section on Page 6 17 E Z N A Plasmid DNA Mini Kit II Spin Protocol Transfer suspension into a new 2 mL microcentrifuge tube Add 500 uL Solution Il Invert and gently rotate the tube several times to obtain a clear lysate A 2 3 minute incubation may be necessary Note Avoid vigorous mixing as this will shear chromosomal DNA and lower plasmid purity Do not allow the lysis reaction to proceed more than 5 minutes Store Solution Il tightly capped when not in use to avoid acidification from CO in the air Add 700 uL Solution Ill Immediately invert several times until a flocculent white precipitate forms Note It is vital that the solution is mixed thoroughly and immediately after the addition of Solution III to avoid localized precipitation Centrifuge at maximum speed 213 000 x g for 10 minutes at room temperature A compact white pellet will form Promptly pr
19. te incubation may be necessary Note Avoid vigorous mixing as this will shear chromosomal DNA and lower plasmid purity Do not allow the lysis reaction to proceed more than 5 minutes Store Solution Il tightly capped when not in use to avoid acidification from CO in the air Add 350 uL Solution Ill Immediately invert several times until a flocculent white precipitate forms Note It is vital that the solution is mixed thoroughly and immediately after the addition of Solution III to avoid localized precipitation Centrifuge at maximum speed 213 000 x g for 10 minutes A compact white pellet will form Promptly proceed to the next step Insert a HiBind DNA Mini Column into a 2 mL Collection Tube Optional Protocol for Column Equilibration 10 11 12 13 14 1 Add 100 uL 3M NaOH to the HiBind DNA Mini Column 2 Centrifuge at maximum speed for 30 60 seconds 3 Discard the filtrate and reuse the collection tube Transfer the cleared supernatant from Step 8 by CAREFULLY aspirating it into the HiBind DNA Mini Column Be careful not to disturb the pellet and that no cellular debris is transferred to the HiBind DNA Mini Column Centrifuge at maximum speed for 1 minute Discard the filtrate and reuse the collection tube Add 500 uL HB Buffer Centrifuge at maximum speed for 1 minute 11 19 16 17 18 E Z N A Plasmid DNA Mini Kit I Spin Protocol Discard the filtrate and reuse collection t
20. tion and purification of nucleic acids from a variety of sources The key to this system is the HiBind matrix that specifically but reversibly binds DNA or RNA under optimized conditions allowing proteins and other contaminants to be removed Nucleic acids are easily eluted with deionized water or low salt buffer The E Z N A Plasmid DNA Mini Kits combine the power of HiBind technology with the time tested consistency of alkaline SDS lysis of bacterial cells to deliver high quality DNA in less than 30 minutes HiBind DNA Mini Columns facilitate the binding washing and elution steps thus enabling multiple samples to be processed simultaneously Typically a 1 5 mL overnight culture in LB medium produces 3 12 ug plasmid DNA although yields may vary according to plasmid copy number E coli strain and growth conditions The E Z N A Plasmid DNA Mini Kit is used to isolate plasmid DNA from 1 5 mL cultures The E Z N A Plasmid DNA Mini Kit II can isolate 40 75 ug plasmid DNA from 10 15 mL cultures when using high copy plasmids Purified plasmid DNA can be directly used for most downstream applications including automated fluorescent DNA sequencing and restriction enzyme digestion New In this Edition The latest edition has been newly designed to enhance readability and protocol quality Equilibration Buffer is no longer included with this kit An optional Column Equilibration Protocol has been added to the protocol for your conven
21. ube Add 700 uL DNA Wash Buffer Note DNA Wash Buffer must be diluted with 10096 ethanol prior to use Please see Page 6 for instructions Centrifuge at maximum speed for 1 minute Discard the filtrate and reuse the collection tube Optional Repeat Steps 16 18 for a second DNA Wash Buffer wash step 19 20 21 22 23 24 12 Centrifuge the empty HiBind DNA Mini Column for 2 minutes at maximum speed to dry the column matrix Note It is important to dry the HiBind DNA Mini Column matrix before elution Residual ethanol may interfere with downstream applications Transfer the HiBind DNA Mini Column to a clean 1 5 mL microcentrifuge tube Add 30 100 uL Elution Buffer or sterile deionized water directly to the center of the column membrane Note The efficiency of eluting DNA from the HiBind DNA Mini Column is dependent on pH If using sterile deionized water make sure that the pH is around 8 5 Let sit at room temperature for 1 minute Centrifuge at maximum speed for 1 minute Note This represents approximately 7096 of bound DNA An optional second elution will yield any residual DNA though at a lower concentration Store DNA at 20 C E Z N A Plasmid DNA Mini Kit I Vacuum Protocol E Z N A Plasmid Mini Kit Protocol Vacuum Protocol All centrifugation should be performed at room temperature unless otherwise noted For low copy number plasmids refer to Page 21 This protocol is designed t
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