E.Z.N.A.® Yeast DNA Kit - Omega Bio-Tek
Contents
1. Prepare the vacuum manifold according to manufacturer s instructions and connect the HiBind DNA Mini Column to the manifold 11 E Z N A Yeast DNA Kit Protocols Optional Protocol for Column Equilibration 17 18 19 20 21 22 23 24 25 26 27 12 1 Add 100 uL 3M NaOH to the HiBind DNA Mini Column 2 Turn on the vacuum source to draw the NaOH through the column 3 Turn off the vacuum Transfer the entire sample from Step 15 to the HiBind DNA Mini Column including any precipitate that may have formed Turn on vacuum source to draw the sample through the column Turn off the vacuum Add 500 uL HBC Buffer to the HiBind DNA Mini Column Note HBC Buffer must be diluted with isopropanol before use Please see Page 4 for instructions Turn on vacuum source to draw the HBC Buffer through the column Turn off the vacuum Add 700 uL DNA Wash Buffer to the HiBind DNA Mini Column Note DNA Wash Buffer must be diluted with ethanol before use Please see Page 4 for instructions Turn on vacuum source to draw the DNA Wash Buffer through the column Turn off the vacuum Repeat Steps 23 25 for a second DNA Wash step Remove the HiBind DNA Mini Column from the vacuum manifold and transfer to a new 2 mL Collection Tube 28 29 30 31 32 33 34 E Z N A Yeast DNA Kit Protocols Centrifuge the empty HiBind DNA Mini Column for 2 minutes at ma2. Any combination of the following steps can be used to help increase DNA yield After adding the Elution Buffer incubate the column for 5 minutes Increase the elution volume Repeat the elution step with fresh Elution Buffer this may increase the yield but decrease the concentration Repeat the elution step using the eluate from the first elution this may increase yield while maintaining elution volume Store DNA at 20 C E Z N A Yeast DNA Kit Protocols E Z N A Yeast DNA Kit Protocol Vacuum Protocol Please read through previous section of this manual before using this protocol Materials and Equipment to be Supplied by User Vacuum manifold for microcentrifuge tubes Vacuum source Tabletop microcentrifuge capable of at least 10 000 x g Nuclease free 1 5 mL microcentrifuge tubes Incubators or water baths capable of at least 65 C Shaking water bath capable of at least 55 C 100 Ethanol Isopropanol B mercatopethanol Optional 3M NaOH Before Starting 10 Prepare the SE Buffer lyticase solution and DNA Wash Buffer according to the instructions in the Preparing Reagents section on Page 4 Set the water baths and or incubators to 30 C and 65 C Set the shaking water bath to 55 C Heat the Elution Buffer to 65 C Culture yeast in YPD medium to an OD of 10 Centrifuge lt 3 mL culture lt 2 x 10 at 4 000 x g for 10 minutes Aspirate and discard the medium Resuspend cells
3. If a precipitate is present heat the bottle at 37 C to dissolve Preparing Reagents 1 Prepare SE Buffer with B mercaptoethanol Add 10 uL B mercaptoethanol per 1 mL SE Buffer The mixture is stable for 1 month at room temperature 2 Prepare a lyticase stock solution at 5 000 units mL and aliquot Store aliquots at 20 C E SE Buffer to be Added D3370 00 220 uL 3 Dilute DNA Wash Buffer with 100 ethanol as follows and store at room temperature ee HopisEthaneitobe dden 4 Dilute HBC Buffer with isopropanol as follows and store at room temperature Recommended Settings The following is required for use with the Vacuum Protocol A Vacuum Manifold We recommend Omega Bio tek s VAC 08 Other Compatible Vacuum Manifolds Qiagen QlAvac24 Sigma Aldrich VM20 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 inHg Tor Tom atmospheres atm pounds per square inch psi Illustrated Vacuum Setup ies N EES oh Feen OMEGA Omega Bio tek s VAC 08 C Vacuum Tubing A Vacuum Manifold D Vacuum Source B Vacuum Flask E Z N A Yeast DNA Kit Protocols E Z N A Yeast DNA Kit Protocol Centrifugati
4. Add 20 uL Proteinase K Solution Vortex to mix well Incubate at 55 C in a shaking water bath to completely lysis the cells Note Usually no more than 1 hour is required for lysis If a shaking water bath is not available incubate and shake or briefly vortex the samples every 20 30 minutes Add 5 uL RNase A Invert several times to mix Let sit at room temperature for 5 minutes Optional Centrifuge at 10 000 x g for 5 minutes to pellet insoluble debris Carefully aspirate the supernatant and transfer to a clean microcentrifuge tube leaving behind any insoluble pellet 13 14 15 16 Add 220 uL YDL Buffer Vortex at maximum speed for 15 seconds Note A wispy precipitate may form upon addition of YDL Buffer it does not interfere with DNA recovery Incubate at 65 C for 10 minutes Add 220 uL 100 ethanol Vortex at maximum speed for 20 seconds If any precipitates can be seen at this point break the precipitates by pipetting up and down 10 times Insert a HiBind DNA Mini Column into a 2 mL Collection Tube E Z N A Yeast DNA Kit Protocols Optional Protocol for Column Equilibration 17 18 19 20 21 22 23 24 25 26 27 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 entire sample from Step 15 to the column including any precipitates that may have
5. in 480 uL SE Buffer and 40 uL lyticase solution Note B mercaptoethanol must be added to the SE Buffer before preparing the lyticase solution Please refer to the Preparing Reagents section on Page 4 for instructions Incubate at 30 C for at least 30 minutes 10 11 12 E Z N A Yeast DNA Kit Protocols Centrifuge at 500 x g for 10 minutes to pellet the spheroblasts Resuspend cells in 200 uL YL Buffer Add 50 mg glass beads Vortex for 5 minutes Add 20 uL Proteinase K Solution Vortex to mix well Incubate at 55 C in a shaking water bath to completely lysis the cells Note Usually no more than 1 hour is required for lysis If a shaking water bath is not available incubate and shake or briefly vortex the samples every 20 30 minutes Add 5 uL RNase A Invert several times to mix Let sit at room temperature for 5 minutes Optional Centrifuge at 10 000 x g for 5 minutes to pellet insoluble debris Carefully aspirate the supernatant and transfer to a clean microcentrifuge tube leaving behind any insoluble pellet 13 14 15 16 Add 220 uL YDL Buffer Vortex at maximum speed for 15 seconds Note A wispy precipitate may form upon addition of YDL Buffer it does not interfere with DNA recovery Incubate at 65 C for 10 minutes Add 220 uL 100 ethanol Vortex at maximum speed for 20 seconds If any precipitates can be seen at this point break the precipitates by pipetting up and down 10 times
6. E Z N A Yeast DNA Kit D3370 00 5 preps D3370 01 50 preps D3370 02 200 preps May 2013 E Z N A Yeast DNA Kit Table of Contents Introduction and OVEFVIEW ssccsscsscsecsseccecseecnseeseecseceneersees 2 Kit Contents Storage and Stability 3 Preparing Regenta 4 Recommended SettiNS ccsccsesssessessseseessecseesssssesseceeeeseeseeses 5 Centrifugation Protocol 6 Vacuum Protocol 10 Troubleshooting Guide onssa ads 14 OA SS 16 Manual Revision May 2013 Fi OMEGA bio tek Innovations in nucleic acid isolation Introduction and Overview Introduction The E Z N A Yeast DNA Kit allows for the rapid and reliable isolation of high quality total cellular DNA from a wide variety of yeast species Up to 3 mL log phase culture OD of 10 in YPD medium can be processed The system combines the reversible nucleic acid binding properties of HiBind matrix with the speed and versatility of spin column technology to yield approximately 15 30 ug DNA with an A A ratio of 1 7 1 9 Purified DNA is suitable for PCR restriction enzyme digestion and hybridization applications There are no organic extractions thus reducing plastic waste and hands on time to allow multiple samples to be processed in parallel Note E Z N A Yeast DNA Kit will isolate all cellular DNA including plasmid DNA Overview If using the E Z N A Yeast DNA Kit for the first time please read this manual before beginning the procedure Yeast
7. at OD Sample too large of 10 or 2x 10 cell per spin column For larger volumes divide sample into multiple tubes Add more lyticase or extend the incubation time It may be necessary to increase the incubation by 60 minutes Repeat elution or increase elution volume Low DNA Poor elution Incubation of column at 65 C for 5 minutes after yield addition of Elution Buffer may increase yields a DNA Wash Buffer must be diluted with 100 Improper washing anal Resin from the column may be present in eluate Extended Avoid centrifugation at speeds higher than centrifugation during specified The material can be removed from the elution step eluate by centrifugation it will not interfere with PCR or restriction digests Incomplete removal of cell wall Incomplete mixing Repeat the procedure this time making sure to with YDL Buffer immediately vortex the sample with YDL Buffer ER i Increase incubation time with YL Buffer Ensure Insufficient incubation u E that no visible cell clumps remain 14 Troubleshooting Guide oven ewe Tan Poor cell lysis due to improper mixing with YDL Buffer Mix thoroughly with YDL Buffer and incubate at 70 C prior to adding ethanol Add more lyticase or extend the incubation time It may be necessary to increase the incubation by 60 minutes Incomplete spheroblasting Ethanol not added to lysate YDL Buffer mixture Ethanol was not added Dilute Wash Buffer with the indica
8. cells are grown to log phase and spheroblasts are subsequently prepared Following lysis binding conditions are adjusted and the sample is applied to a HiBind DNA Mini Column Three rapid wash steps remove trace salts and protein contaminants DNA is eluted in water or low ionic strength buffer Purified DNA can be directly used in downstream applications without the need for further purification New in this Edition HB Buffer has been replaced by HBC Buffer Isopropanol is required and supplied by the user Equilibration Buffer is no longer included with this kit An optional Column Equilibration Protocol has been added to the protocol for your convenience Equilibration Buffer is replaced with 3M NaOH provided by the user Kit Contents D3370 00 D3370 01 2 mL Collection Tubes YL Buffer YDL Buffer HBC Buffer DNA Wash Buffer Glass Beads Elution Buffer SE Buffer Lyticase Proteinase K Solution RNase A User Manual YDL Buffer contains a chaotropic salt Storage and Stability All components of the E Z N A Yeast DNA Kit except the RNase A and lyticase can be stored at 22 25 C and are guaranteed for at least 12 months from the date of purchase Proteinase K Solution can be stored at room temperature for 12 months For long term storage gt 12 months store Proteinase K Solution at 2 8 C Store RNase A at 20 C Under cool ambient conditions a precipitate may form in the YL Buffer and or YDL Buffer
9. formed Centrifuge at 10 000 x g for 1 minute Discard the filtrate and the Collection Tube Insert the HiBind DNA Mini Column into a new 2 mL Collection Tube Add 500 uL HBC Buffer Note HBC Buffer must be diluted with isopropanol before use Please see Page 4 for instructions Centrifuge at 10 000 x g for 1 minute Discard the filtrate and reuse the Collection Tube Add 700 uL DNA Wash Buffer Note DNA Wash Buffer must be diluted with 100 ethanol according to the instructions in the Preparing Reagents section on Page 4 Centrifuge at 10 000 x g for 1 minute Discard the filtrate and reuse the Collection Tube Repeat Steps 24 26 for a second DNA Wash Buffer wash step 28 29 30 31 32 33 34 E Z N A Yeast DNA Kit Protocols Centrifuge the empty HiBind DNA Mini Column for 2 minutes at maximum speed 210 000 x g to dry the column matrix Note It is important to dry the column membrane before elution Residual ethanol may interfere with downstream applications Transfer the HiBind DNA Mini Column into a nuclease free 1 5 mL microcentrifuge tube Add 50 100 uL Elution Buffer heated to 65 C Let sit at room temperature for 3 to 5 minutes Note Incubating the HiBind DNA Mini Column at 65 C rather than room temperature will give a modest increase in DNA yield per elution Centrifuge at 10 000 x g for 1 minute Repeat Steps 30 32 for a second elution step Note
10. on Protocol This method isolates genomic DNA from up to 3 mL yeast culture lt 2 x 10 cells All centrifugation steps should be performed at room temperature Materials and Equipment to be Supplied by User Tabletop microcentrifuge capable of at least 10 000 x g Nuclease free 1 5 mL microcentrifuge tubes Incubators or water baths capable of at least 65 C Shaking water bath capable of at least 55 C 100 Ethanol e Isopropanol B mercatopethanol Optional 3M NaOH Before Starting Prepare the SE Buffer lyticase solution and DNA Wash Buffer according to the instructions in the Preparing Reagents section on Page 4 Set the water baths and or incubators to 30 C and 65 C Set the shaking water bath to 55 C Heat the Elution Buffer to 65 C 1 Culture yeast in YPD medium to an OD of 10 2 Centrifuge lt 3 mL culture lt 2 x 10 at 4 000 x g for 10 minutes 3 Aspirate and discard the medium 4 Resuspend cells in 480 uL SE Buffer and 40 uL lyticase solution Note B mercaptoethanol must be added to the SE Buffer before preparing the lyticase solution Please refer to the Preparing Reagents section on Page 4 for instructions 5 Incubate at 30 C for at least 30 minutes 10 11 12 E Z N A Yeast DNA Kit Protocols Centrifuge at 500 x g for 10 minutes to pellet the spheroblasts Resuspend cells in 200 uL YL Buffer Add 50 mg glass beads Vortex for 5 minutes
11. ted volume of to DNA Wash Buffer ethanol before use Before applying sample to column an aliquot of ethanol must be added See protocol above 15 16 Ordering Information The following components are available for purchase separately Call Toll Free at 1 800 832 8896 Vacuum Manifold VAC 08 1 5 mL DNase RNase free Microcentrifuge Tubes 1000 pk 10 pk cs SSI 1210 00 YL Buffer 100 mL PDO91 DNA Wash Buffer 100 mL PSO10 Elution Buffer 100 mL PDRO48 RNase A 25 mg mL 400 uL AC117 HiBind E Z N A and MicroElute are registered trademarks of Omega Bio tek Inc Qiagen QlAvac 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
12. ximum speed 210 000 x g to dry the column matrix Note It is important to dry the column membrane before elution Residual ethanol may interfere with downstream applications Transfer the HiBind DNA Mini Column into a nuclease free 1 5 mL microcentrifuge tube Add 50 100 uL Elution Buffer heated to 65 C Let sit at room temperature for 3 to 5 minutes Note Incubating the HiBind DNA Mini Column at 65 C rather than room temperature will give a modest increase in DNA yield per elution Centrifuge at 10 000 x g for 1 minute Repeat Steps 30 32 for a second elution step Note Any combination of the following steps can be used to help increase DNA yield After adding the Elution Buffer incubate the column for 5 minutes Increase the elution volume Repeat the elution step with fresh Elution Buffer this may increase the yield but decrease the concentration Repeat the elution step using the eluate from the first elution this may increase yield while maintaining elution volume Store DNA at 20 C 13 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 oven ewe Tann Add the correct volume of YL Buffer and incubate at 55 C to obtain complete lysis It may be necessary to extend incubation time to 30 minutes Incomplete lysis Do not use greater than 3 mL culture
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