Manual - Omega Bio-Tek
Contents
1. eluted with deionized water or a low salt buffer DNA and RNA are suitable for many downstream applications Omega Bio tek s E Z N A M13 DNA Kits are designed to purify up to 10 ug of single stranded DNA from up to 3 mL of phage supernatant Yields of single stranded DNA obtained using E Z N A M13 DNA Kits are around 3 10 ug and reproducible when the isolations are performed from the same culture Overview The E Z N A M13 DNA Kit isolation procedures first call for the infected bacterial culture to be centrifuged to pellet the bacterial cells MPG buffer is added to the supernatant to precipitate the phage particles The samples are loaded on HiBind M13 DNA Mini Columns or on to E Z 96 DNA Plates The specially designed HiBind matrix will retain intact phage particles These phage particles are lysed and bound to the HiBind matrix after the addition of MPX Buffer Contaminants such as protein are efficiently washed away with SPW Wash Buffer and pure ssDNA is eluted with Elution Buffer New in this Edition This manual has been edited for content and redesigned to enhance user readability Kit Contents rods ooo oeoo sewel SquarewellPates 22m 1 a Storage and Stability All E Z N A or E Z 96 M13 DNA Kit components are guaranteed for at least 12 months from the date of purchase when stored at room temperature Preparing Reagents 1 Dilute SPW Wash Buffer with 100 ethanol as follows and store at roo2. the buffer passes through the well membranes E Z 96 DNA Kit Protocols 20 Turn off the vacuum source and vent the manifold 21 Repeat Steps 18 20 for a second SPW Wash Buffer wash step 22 Remove the E Z 96 DNA Plate from the vacuum manifold 23 Strike the bottom of the E Z 96 DNA Plate on a stack of paper towels Repeat a few times until there is no liquid released onto the paper towels 24 Place the E Z 96 DNA Plate back to the top plate of the manifold 25 Apply the vacuum for 5 minutes 26 Turn off the vacuum source and vent the manifold 27 Remove the E Z 96 DNA Plate from the vacuum manifold 28 Strike the bottom of the E Z 96 DNA Plate on a stack of paper towels Repeat a few times until there is no liquid released onto the paper towels Optional Place the E Z 96 DNA Plate into a vacuum oven preset at 65 C and incubate for 10 minutes to completely dry the plate 29 Replace the waste collection tray with a set of 96 well Racked Microtubes 1 2 mL 30 Reassemble the manifold with the E Z 96 DNA Plate on top of the manifold 31 Add 75 150 uL Elution Buffer heated to 65 C Note Make sure to add the Elution Buffer directly onto the E Z 96 DNA Plate matrix 32 Let sit for 10 minutes at room temperature 13 33 34 35 36 37 14 E Z 96 DNA Kit Protocols Apply the vacuum for 5 minutes Turn off the vacuum and vent the manifold Disassemble the vacuum manifold Seal th
3. Ca OMEGA Innovations in nucleic acid isolation Product Manual E Z N A M13 DNA Mini Kit D6900 00 5 preps D6900 01 50 preps EZ 96 M13 DNA Kit D1900 00 2 preps D1900 01 6 preps June 2013 For research use only Not intended for diagnostic testing E Z N A M13 DNA Mini Kit E Z 96 M13 DNA Kit Table of Contents Introduction and OvervieW ssssssssssssssssseessssssseseeseeseeseeeeees 2 Kit Contents Storage and Stability ssesssssssecseeseees 3 Preparing Reagents sseessrssssrsreeesssseseessssseceessseeecossssesosssseees 4 E Z N A M13 DNA Mini PROCGCOl scssscsssscarsseieisessieanseccereans 5 E Z 96 M13 DNA Spin Protocol ssssssssssessssssssossessessseeeereeees 8 E Z 96 M13 DNA Vacuum Protocol cssessesssesseeseecsssseesees 11 Troubleshooting Guide sesssesseessessssssssseeessesssssssseeeseesensssss 15 O Orderifgsestsosssnunrunene nan ain aas 16 Manual Revision June 2013 02 OMEGA bio tek Innovations in nucleic acid isolation Introduction and Overview Introduction The E Z N A family of products is an innovative system that radically simplifies the extraction and purification of nucleic acids from a variety of sources The key to this system is Omega Bio tek s HiBind matrix that specifically but reversibly binds DNA or RNA under optimal conditions while allowing proteins and other contaminants to be removed The nucleic acids bound to the HiBind matrix are easily
4. e 96 well Racked Microtubes with the Caps for Racked Microtubes Store eluted DNA at 20 C 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 1 800 832 8896 Problem Low DNA yields Incorrect host stain Bacterial culture overgrown or not fresh Lower pH on the elution buffer Elution buffer did not cover the membrane completely Column clogged Make sure that host strain carries the F episome which is essential for M13 infection Do not incubate cultures for more than 8 hr at 37 C Make sure the pH of the elution solution is between 7 5 8 0 Make sure that elution buffer is dispensed directly onto the center of the membrane Use less than 3 mL M13 phage supernatant per column Avoid the bacterial pellet during transfer paa fe ea No DNA eluted High molecular weight DNA contamination of product Optical densities do not agree with DNA yield on agarose gel DNA floats out of well while loading agarose gel SPW Wash Buffer not diluted with ethanol Carryover of the bacterial cell during transfer Trace contaminants eluted from column increase A260 Ethanol not completely removed from column following wash steps Prepare SPW Wash Buffer as instructed on Page 4 Make sure not to carry any bacteria during the transfer of the supernatant An extra ce
5. e filtrate and reuse the collection tube Add 700uL MPX Buffer Let sit for 1 minute at room temperature Centrifuge at 10 000 rpm for 30 seconds Discard the filtrate and reuse the collection tube Add 700 uL SPW Wash Buffer Note SPW Wash Buffer must be diluted with ethanol before use Please see Page 4 for instructions Centrifuge at 10 000 rpm for 30 seconds Discard the filtrate and reuse the Collection Tube Repeat Steps 17 19 for a second SPW Wash Buffer wash step Centrifuge the empty column at maximum speed for 1 minute 22 23 24 25 26 E Z N A M13 Mini Protocol Insert the HiBind M13 DNA Mini Column into a clean 1 5 mL microcentrifuge tube not provided Add 50 100 uL Elution Buffer heated to 65 C Note Make sure to add Elution Buffer directly onto the HiBind M13 DNA Mini Column matrix Let sit at room temperature for 10 minutes Centrifuge at maximum speed for 1 minute Store eluted DNA at 20 C E Z 96 DNA Kit Protocols E Z 96 M13 DNA Kit Protocol Spin Protocol Materials and Equipment to be Supplied by User 100 Ethanol e Centrifuge capable of at least 3 000 x g e Centrifuge adaptor for 96 well plates Water bath or incubator capable of 65 C Sealing film Before Starting Prepare SPW Wash Buffer according to the instructions on Page 4 Heat the Elution Buffer to 65 C 1 Prepare an infected M13 culture grown in a 2 2 mL 96 well cultur
6. e plate not supplied 2 Centrifuge at 5 000 rpm for 15 minutes at room temperature 3 Transfer 1 2 mL supernatant to a 2 mL Collection Plate Note Be careful not to disturb the bacterial pellet during the transfer If the supernatant is not clear repeat the centrifugation step 4 Add 1 5 volume MPG Buffer 200 uL MPG per 1 mL culture to the M13 supernatant Vortex to mix thoroughly 5 Letsit for 15 minutes at room temperature 6 Place the E Z 96 DNA Plate on top of a 96 well Square well Plate 2 2 mL 7 Transfer 1 mL supernatant to the E Z 96 DNA Plate 10 11 12 13 14 15 16 17 18 19 20 21 E Z 96 DNA Kit Protocols Centrifuge at 3 000 x g for 5 minutes Discard the filtrate and reuse the 96 well Square well Plate Repeat Steps 7 9 until all of the cleared supernatant has been transfer to the E Z 96 DNA Plate Add 1 mL MPX Buffer Centrifuge at 3 000 x g for 5 minutes Discard the filtrate and reuse the 96 well Square well Plate Add 1 mL MPX Buffer Let sit for 1 minute at room temperature Centrifuge at 3 000 x g for 5 minutes Discard the filtrate and reuse the 96 well Square well Plate Place the E Z 96 DNA Plate on top of the 96 well Square well Plate Add 1 mL SPW Wash Buffer Note SPW Wash Buffer must be diluted with ethanol before use Please see Page 4 for instructions Centrifuge at 3 000 x g for 15 minutes Discard the filtrate and reuse the 96 we
7. ll Square well Plate E Z 96 DNA Kit Protocols Optional Place the E Z 96 DNA Plate into a vacuum oven preset at 65 C and incubate for 10 minutes to completely dry the plate 22 23 24 25 26 27 28 10 Place the E Z 96 DNA Plate on top of a set of 96 well Racked Microtubes 1 2 mL Add 75 150 uL Elution Buffer heated to 65 C Note Make sure to add the Elution Buffer directly onto the E Z 96 DNA Plate matrix Let sit for 10 minutes at room temperature Centrifuge at 3 000 x g for 5 minutes Discard the E Z 96 DNA Plate Seal the 96 well Racked Microtubes with the Caps for Racked Microtubes Store eluted DNA at 20 C E Z 96 DNA Kit Protocols E Z 96 M13 DNA Kit Protocol Vacuum Protocol Materials and Equipment to be Supplied by User 100 Ethanol Vacuum manifold for 96 well plates Cat VAC 03 Vacuum source capable of generating a vacuum pressure of 900 mbar Centrifuge with rotor for 96 well plates capable of at least 3 000 x g Water bath or incubator capable of 65 C 96 well culture plates Sealing film Optional Vacuum oven Before Starting Prepare SPW Wash Buffer according to the instructions on Page 4 Heat the Elution Buffer to 65 C Prepare an infected M13 culture grown in a 2 2 mL 96 well culture plate not supplied Centrifuge at 5 000 rpm for 15 minutes at room temperature Transfer 1 2 mL supernatant to a 2 mL Collection Plate Note Be careful not to di
8. m temperature D1900 00 200 mL D1900 01 800 mL E Z N A M13 Mini Protocol E Z N A M13 Miniprep Kit Protocol Materials and Equipment to be Supplied by User 100 Ethanol Microcentrifuge capable of at least 12 000 x g Water bath or incubator capable of 65 C Nuclease free 1 5 mL or 2 0 mL microcentrifuge tubes Before Starting Prepare SPW Wash Buffer according to the instructions on Page 4 Heat the Elution Buffer to 65 C Prepare a 4 mL culture of infected M13 Incubate at 37 C for 6 7 hours with vigorous shaking Centrifuge at 5 000 rpm for 15 minutes at room temperature Transfer 1 4 mL of the supernatant obtained containing the M13 bacteriophage into a fresh reaction tube Note Be careful not to disturb the bacterial pellet during the transfer If the supernatant is not clear repeat the centrifugation step Add 280 uL MPG Buffer to the M13 supernatant and mix by vortexing Let sit at room temperature for 10 15 minutes Add 700 uL sample to a HiBind M13 DNA Mini Column inserted into a 2 mL Collection Tube 10 11 12 13 14 13 16 IZ 18 19 20 21 E Z N A M13 Mini Protocol Centrifuge at 10 000 rpm for 30 seconds Discard the filtrate and reuse the collection tube Repeat Steps 7 and 8 until all of the sample has been passed through the HiBind M13 DNA Mini Column Add 700 uL MPX Buffer Centrifuge for 30 seconds at 10 000 rpm Discard th
9. ntrifugation step may be necessary Make sure to wash column as instructed and rely on agarose gel ethidium bromide electrophoresis for quantitation Centrifuge column as instructed in Step 19 to dry 15 16 Ordering Information The following components are available for purchase separately Call Toll Free at 1 800 832 8896 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 E Z 96 Vacuum Manifold VAC 03 SPW Wash Buffer 25 mL PDRO45 Elution Buffer 100 mL PDRO48 HiBind E Z N A and MicroElute are registered trademarks of Omega Bio tek Inc PCR is a patented process of Hoffman La Roche Use of the PCR process requires a license
10. sturb the bacterial pellet during the transfer If the supernatant is not clear repeat the centrifugation step Add 1 5 volume MPG Buffer 200 uL MPG per 1 mL culture to the M13 supernatant Vortex to mix thoroughly Let sit for 15 minutes at room temperature Assemble the vacuum manifold according to the manufacturer s instructions Note If using the VAC 03 vacuum manifold place the waste collection tray inside the manifold and place the E Z 96 DNA Plate on top of the manifold 11 10 11 12 13 14 15 16 17 18 19 12 E Z 96 DNA Kit Protocols Transfer 1 mL cleared supernatant to the E Z 96 DNA Plate Seal the unused wells with sealing film not provided Apply the vacuum until all the cleared supernatant pass through the well membranes Turn off the vacuum source and vent the manifold Repeat Steps 7 9 until all of the cleared supernatant has been transfer to the E Z 96 DNA Plate Add 1 mL MPX Buffer Immediately apply the vacuum until all the buffer passes through the well membranes Turn off the vacuum source and vent the manifold Add 1 mL MPX Buffer Let sit for 2 minutes at room temperature Apply the vacuum until all the buffer passes through the well membranes Turn off the vacuum source and vent the manifold Add 1 mL SPW Wash Buffer Note SPW Wash Buffer must be diluted with ethanol before use Please see Page 4 for instructions Apply the vacuum until all
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