E.Z.N.A. MicroElute Clean-up Kits Table of Contents

Transcription

1 E.Z.N.A. MicroElute Clean-up Kits Table of Contents Introduction... 2 Kit Contents... 3 Preparing Reagents/Storage and Stability... 4 Guideline for Vacuum Manifold... 5 MicroElute Cycle-Pure - Spin Protocol... 6 MicroElute Cycle-Pure - Vacuum/ Spin Protocol... 8 MicroElute Gel Extraction - Spin Protocol... 9 MicroElute Gel Extraction - Vacuum/ Spin Protocol MicroElute DNA Clean-up - Spin Protocol MicroElute DNA Clean-up - Vacuum/ Spin Protocol Troubleshooting Guide...15 Ordering...16 Manual Revision: July 2010 Innovations in nucleic acid isolation 1

2 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 the new HiBind matrix that specifically, but reversibly, binds DNA or RNA under certain optimal conditions allowing proteins and other contaminants to be removed. Nucleic acids are easily eluted with deionized water or a low salt buffer. The MicroElute Clean-up system, designed for rapid DNA cleanup, includes: MicroElute Cycle-Pure Kit - for direct purification of double or single stranded PCR products (100 bp - 10 kb) from amplification reactions. MicroElute Gel Extraction Kit - for extraction of DNA fragments (70 bp - 20 kb) from standard, or low-melt agarose gels in TAE (Tris-acetate/EDTA) or TBE (Tris-borate/ EDTA) buffer. MicroElute DNA Cleanup Kit - for general cleanup of oligonucleotides and DNA up to 10 kb from enzymatic reactions (e.g., labeling, dephosphorylation, restriction, and tailing). Binding Capacity Each MicroElute HiBind DNA Mini column can bind ~10 µg of DNA. 2

3 Kit Contents MicroElute DNA Cycle-Pure Kit D D D Purification Times 5 preps 50 preps 200 preps MicroElute HiBind DNA Columns ml Collection Tubes CP Buffer 5 ml 30 ml 120 ml Equilibration Buffer 1.5 ml 7 ml 25 ml Elution Buffer 1 ml 15 ml 15 ml DNA Wash Buffer 1.5 ml 15 ml 3 x25 ml Instruction Booklet MicroElute DNA Gel Extraction Kit D D D Purification Times 5 preps 50 preps 200 preps MicroElute HiBindDNA Columns ml Collection Tubes Equilibration Buffer 1.5 ml 7 ml 25 ml Binding Buffer (XP2) 5 ml 30 ml 120 ml Elution Buffer 1 ml 15 ml 15 ml SPW Wash Buffer 2 ml 25 ml 3 x 25 ml Instruction Booklet MicroElute DNA Clean-up Kit D D D Purification Times 5 preps 50 preps 200 preps MicroElute HiBind DNA Columns ml Collection Tubes DP Buffer 2 ml 20 ml 80 ml Equilibration Buffer 1.5 ml 7 ml 25 ml Elution Buffer 1 ml 15 ml 15 ml SPW Wash Buffer 2 ml 25 ml 3 x 25 ml Instruction Booklet

4 Preparing Reagents DNA Wash Buffer and SPW Wash Buffer must be diluted with absolute ethanol (96-100%) as follows: DNA Wash Buffer: Kit Ethanol Added D Add 6 ml absolute ethanol D Add 60 ml absolute ethanol D Add 100 ml absolute ethanol to each bottle SPW Wash Buffer: Kit Ethanol Added D Add 8 ml absolute ethanol D Add 100 ml absolute ethanol D Add 100 ml absolute ethanol to each bottle Kit Ethanol Added D Add 8 ml absolute ethanol D Add 100 ml absolute ethanol D Add 100 ml absolute ethanol to each bottle Storage and Stability All MicroElute Cycle-Pure Kit components are guaranteed for at least 12 months from the date of purchase when stored at C. Under cool ambient conditions, crystals may form in the CP Buffer. Simply warm CP Buffer to 37 C to dissolve. 4

5 Guideline 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 QIAvac24, 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 to -600 Conversion from millibars: Multiply by: Millimeters of mercury (mm Hg) 0.75 Kilopascals (kpa) 0.1 Inches of mercury (inch Hg) Torrs (Torr) 0.75 Atmospheres (atmos) Pounds per Square Inch (psi) Illustrated Vacuum Setup: Omega Bio-tek s VAC-08 C) Vacuum Tubing A) Vacuum Manifold D) Vacuum Source B) Vacuum Flask 5

6 MicroElute Cycle-Pure - Spin Protocol MicroElute Cycle-Pure Spin Protocol User Supplied Equipment: Microcentrifuge capable of at least 13,000 x g Absolute ethanol (~ %) Deionized water 1.5 ml microcentrifuge tube Vortex Before starting: Prepare DNA Wash Buffer according to Preparing Reagents section on page Perform agarose gel/ethidium bromide electrophoresis to analyze PCR product. 2. Determine the volume of the PCR reaction. Transfer the sample into a clean 1.5ml microcentrifuge tube and add 5 volumes of CP Buffer. Note: Volumes refers to the size of your PCR reaction. For example, if your PCR reaction is 50 µl you would use 250 µl of CP Buffer. 3. Vortex thoroughly to mix. Briefly spin the tube to collect any drops from the inside of the lid. 4. Place a HiBind DNA Mini Column into a provided 2 ml collection tube. 5. Add 100 µl of Equilibration to the MicroElute DNA Column. 6. Centrifuge at 10,000 x g for 30 seconds. 7. Add 500 µl of Deionized Water to the MicroElute DNA Column. 8. Centrifuge at 10,000 x g for 30 seconds. Discard the Flow through. 9. Apply the sample to a MicroElute HiBind DNA Column and centrifuge at 10,000 x g for 1 min at room temperature. 10. Discard flow through liquid and reuse the collection tube. 11. Add 700 μl of DNA Wash Buffer diluted with absolute ethanol to the MicroElute HiBind DNA Column. Centrifuge at 10,000 x g for 1 min at room temperature. Discard liquid and re-use collection tube. 6

7 MicroElute Cycle-Pure - Spin Protocol Note: DNA Wash Buffer must be diluted with absolute ethanol before use. If refrigerated, DNA Wash Buffer must be brought to room temperature before use. 12. Add 700 μl of DNA Wash Buffer diluted with absolute ethanol to the MicroElute HiBind DNA Column. Centrifuge at 10,000 x g for 1 min at room temperature. Discard liquid and re-use collection tube. 13. Centrifuge the empty MicroElute HiBind column for 2 min at maximum speed ( 13,000 x g) to dry the column matrix. Important: Do not skip this step, it is critical for good yields. 14. Place MicroElute HiBind DNA column into a clean 1.5 ml microcentrifuge tube(not supplied). Add μl (depending on desired concentration of final product) of Elution Buffer (10mM Tris, ph8.5) or water directly onto the center of the column matrix and incubate at room temperature for 1-2 minutes. Centrifuge for 1 min at 13,000 x g to elute DNA. This represents approximately 80-90% of bound DNA. An optional second elution will yield any residual DNA, though at a lower concentration. 7

8 MicroElute Cycle-Pure - Vacuum/Spin Protocol MicroElute Cycle-Pure - Vacuum/Spin Protocol User Supplied Equipment: Microcentrifuge capable of at least 13,000 x g Absolute ethanol (~ %) Vacuum Manifold with standard luer connector (see page 5) Sterile deionized water 1.5 ml microcentrifuge tube Vacuum Manifold Before starting: Prepare DNA Wash Buffer according to Preparing Reagents section on page Prepare the sample by following steps 1-4 of the spin protocol on page Prepare the vacuum manifold according to manufacturer s instructions. Insert the column into the vacuum leur connector. 3. Add 100 μl of Equilibration Buffer into the column. Turn on the vacuum source to draw liquid through the membrane. Add 500 μl of water into the column and wait until all the liquid passes through the membrane. Turn off the vacuum source. 4. Load the PCR reaction/cp solution to the MicroElute HiBind DNA column by decanting or pipetting, and turn on the vacuum source. After the samples have passed through the column, switch off the vacuum source. 5. Add 700 μl of DNA Wash Buffer diluted with absolute ethanol to the Hibind MicroElute Column and turn on the vacuum source. When the buffer has passed through the column, add an additional 700µL DNA Wash buffer to the HiBind MicroElute Column. When the buffer has passed through the column, turn off the vacuum source. 6. Assemble the MicroElute HiBind DNA Column into a 2 ml collection tube. Spin for 2 min at maximum speed ( 13,000 x g) to dry the MicroElute HiBind DNA column. Important: Do not skip this step, it is critical for good yields. 7. Place the MicroElute HiBind DNA Column in a clean 1.5 ml microcentrifuge tube(not supplied) and add μl of Elution Buffer. Incubate at room temperature for 1-2 minutes. Centrifuge for 1 min at 13, 000 x g to elute DNA. 8

9 MicroElute Gel Extraction Kit - Spin Protocol MicroElute Gel Extraction Kit - Spin Protocol User Supplied Equipment: Microcentrifuge capable of at least 13,000 x g Absolute ethanol (~ %) deionized water 1.5 ml microcentrifuge tube Incubator capable of 55 C Vortex Before starting: Prepare SPW Wash Buffer according to Preparing Reagents section on page 4. Set an incubator to 55 C 1. Perform agarose gel/ethidium bromide electrophoresis to fractionate DNA fragments. Any type or grade of agarose may be used. It is strongly recommended however, that fresh TAE buffer, or TBE buffer be used as running buffer. Do not reuse running buffer as its ph will increase and reduce yields. 2. When adequate separation of bands has occurred, carefully excise the DNA fragment of interest using a UV light box ensuring that as much agarose gel as possible has been removed. Avoid more than 30 seconds of exposure of UV light to the DNA. Always use protective eye-ware when working with UV light. 3. Determine the appropriate volume of the gel slice by weighing it in a clean 1.5 ml microcentrifuge tube. Assuming a density of 1g/ml of gel, the volume of gel is derived as follows: a gel slice of mass 0.2g will have a volume of 0.2 ml. Add an equal volume of Binding Buffer. Incubate the mixture at C for 7 min or until the gel has completely melted. Mix by shaking or vortexing the tube in increments of 2-3 minutes. Note: Monitor the ph of the Gel/Binding Buffer mixture after the gel has completely dissolved. DNA yields will significantly decrease when ph > 8.0. If the color of the mixture becomes orange or red, add 5 µl of 5M sodium acetate, ph 5.2 to bring the ph down. After this adjustment, the color of the Gel/Binding Buffer mixture should be light yellow. 4. Place a MicroElute HiBind DNA Column in a provided 2 ml collection tube 5. Add 100 µl of Equilibration Buffer to the MicroElute DNA Column. 6. Centrifuge at 10,000 x g for 30 seconds. 7. Add 500 µl of Deionized Water to the MicroElute DNA Column. 9

10 MicroElute Gel Extraction Kit - Spin Protocol 8. Centrifuge at 10,000 x g for 30 seconds. Discard the Flow through. 9. Apply the DNA/agarose solution (no more than 700 μl at a time) to the MicroElute HiBind DNA Column, and centrifuge at 10, 000 x g for 1 min at room temperature. 10. Discard liquid and place the MicroElute HiBind DNA Column back into the same collection tube. For volumes greater than 700 μl, load the column and centrifuge successively, 700 µl at a time. Each MicroElute HiBind DNA column has a total capacity of 10 µg DNA. If the expected yield is larger, divide the sample into the appropriate number of columns. 11. Add 300 µl of Binding Buffer (XP2) into the MicroElute HiBind DNA Column. Centrifuge at 13,000 x g for 30 seconds to wash the column. Discard the flow-through and re-use the collection tube. 12. Add 700 μl of SPW Wash Buffer to the MicroElute Hibind DNA Column. Centrifuge at 13,000 x g for 30 seconds and discard the flow-through and reuse the collection tube. Note: SPW Wash Buffer must be diluted with absolute ethanol before use. See label for directions. 13. Optional: Repeat step 12 with another 700 μl of SPW Wash Buffer. 14. Centrifuge the empty MicroElute HiBind DNA Column for 2 min at maxi speed ( 13,000 x g) to dry the column matrix. This is critical for good yields. Important: Do not skip this step, it is critical for good yields. 15. Place MicroElute HiBind DNA Column into a clean 1.5 ml microcentrifuge tube. Add μl (depending on desired concentration of final product) of Elution Buffer (10mM Tris, ph8.5 ) or water directly onto the column matrix and let it sit at room temperature for 1-2 minutes. Centrifuge for 1 min at 13,000 x g to elute DNA. This represents approximately 70-80% of bound DNA. An optional second elution will yield any residual DNA, though at a lower concentration. 10

11 MicroElute Gel Extraction Kit - Vacuum/Spin Protocol MicroElute Gel Extraction Kit - Vacuum/Spin Protocol User Supplied Equipment: Microcentrifuge capable of at least 13,000 x g Absolute ethanol (~ %) deionized water 1.5 ml microcentrifuge tube Incubator capable of 55 C Vortex Vacuum Manifold Before starting: Prepare SPW Wash Buffer according to Preparing Reagents section on page 4. Set an incubator to 55 C 1. Prepare the sample by following steps 1-3 of the spin protocol on page Prepare the vacuum manifold according to manufacturer s instructions. Insert the column into the vacuum leur connector. 3. Add 100 μl of Equilibration Buffer into the column. Turn on the vacuum source to draw liquid through the membrane. Add 500 μl of water into the column and wait until all the liquid passes through the membrane. Turn off the vacuum source. 4. Load the DNA/Agarose solution to the MicroElute HiBind DNA column by decanting or pipetting, and turn on the vacuum source. After the liquid has passed through the column, switch off the vacuum source. 5. Add 300 μl of Binding Buffer (XP2) and on the vacuum source. When the Buffer has passed through the column, turn off the vacuum source. 6. Add 700 μl of SPW Wash Buffer diluted with absolute ethanol to the Hibind MicroElute Column and turn on the vacuum source. When the buffer has passed through the column, add an additional 700µL SPW Wash buffer to the HiBind MicroElute Column. When the buffer has passed through the column, turn off the vacuum source. 7. Assemble the MicroElute HiBind DNA Column into a 2 ml collection tube(supplied). Spin for 2 min at maximum speed ( 13,000 x g) to dry the MicroElute HiBind DNA column. Important: Do not skip this step, it is critical for good yields. 8. Place the MicroElute HiBind DNA Column in a clean 1.5 ml microcentrifuge tube(not supplied) and add μl of Elution Buffer. Incubate at room temperature for 1-2 minutes. Centrifuge for 1 min at 13, 000 x g to elute DNA. 11

12 MicroElute DNA Clean-up Kit - Spin Protocol MicroElute DNA Clean-up Kit - Spin Protocol User Supplied Equipment: Microcentrifuge capable of at least 13,000 x g Absolute ethanol (~ %) Sterile deionized water 1.5 ml microcentrifuge tube Vortex Before starting: Prepare SPW Wash Buffer according to Preparing Reagents section on page Determine the volume of the enzymatic reaction, transfer sample into a clean 1.5 ml microcentrifuge tube, and add 3 volumes of Buffer DP. Vortex thoroughly to mix. For example, add 300 μl of DP Buffer to 100 μl enzymatic reaction. 2. Place a MicroElute HiBindDNA Column in a provided 2 ml collection tube. 3. Add 100 µl of Equilibration to the MicroElute DNA Column. 4. Centrifuge at 10,000 x g for 30 seconds. 5. Add 500 µl of Deionized Water to the MicroElute DNA Column. 6. Centrifuge at 10,000 x g for 30 seconds. Discard the Flow through. 7. Apply the sample to the MicroElute HiBind DNA Column, and centrifuge at 10,000 x g for 1 min at room temperature to bind DNA. 8. Discard liquid and place the MicroElute HiBind DNA Column back into the same collection tube. 9. Add 700 μl of SPW Wash Buffer to the MicroElute Hibind DNA Column. Centrifuge at 13,000 x g for 30 seconds and discard the flow-through and re-use the 2 ml collection tube in the next step. Note: SPW Wash Buffer must be diluted with absolute ethanol before use. See label for directions. 12

13 MicroElute DNA Clean-up Kit - Spin Protocol 10. Add 700 μl of SPW Wash Buffer to the MicroElute Hibind DNA Column. Centrifuge at 13,000 x g for 30 seconds and discard the flow-through and re-use the 2 ml collection tube in the next step. 11. Centrifuge the empty MicroElute HiBind DNA Column for 2 min at maximum speed ( 13,000 x g) to dry the column matrix. This is critical for good yields. Important: Do not skip this step, it is critical for good yields. 12. Place MicroElute HiBind DNA column into a clean 1.5 ml microcentrifuge tube(not supplied). Add μl of Elution Buffer (10mM Tris, ph8.5) directly onto the column matrix and incubate at room temperature for 1 min. 13. Centrifuge for 1 min at 13,000 x g to elute DNA. This represents approximately 80-90% of bound DNA. An OPTIONAL second elution will yield any residual DNA, though at a lower concentration. 13

14 MicroElute DNA Clean-up Kit - Vacuum/Spin Protocol MicroElute DNA Clean-up Kit - Vacuum/Spin Protocol User Supplied Equipment: Microcentrifuge capable of at least 13,000 x g Absolute ethanol (~ %) Sterile deionized water 1.5 ml microcentrifuge tube Vortex Vacuum Manifold Before starting: Prepare SPW Wash Buffer according to Preparing Reagents section on page Determine the volume of the enzymatic reaction, transfer sample into a clean 1.5 ml microcentrifuge tube, and add 3 volumes of DP Buffer. Vortex thoroughly to mix For example, add 300 μl of Buffer DP to 100 μl enzymatic reaction. 2. Prepare the vacuum manifold according to manufacturer s instructions, and connect the MicroElute HIBind DNA Column to the manifold. 3. Add 100 µl of Equilibration Buffer to the MicroElute DNA Column. Turn on the vacuum source and let the liquid flow through. 4. Add 500 µl of Deionized Water to the MicroElute DNA Column. Turn on the vacuum source and let the liquid flow through. 5. Load the sample to the MicroElute HiBind DNA column by decanting or pipetting, and turn on the vacuum source. After the liquid has passed through the column, switch off the vacuum source. 6. Add 700 μl of SPW Wash Buffer diluted with absolute ethanol to the MicroElute HiBind DNA Column and turn on the vacuum source. When the buffer has passed through the column, add an additional 700µL SPW Wash buffer to the HiBind MicroElute Column. When the buffer has passed through the column, turn off the vacuum source. 7. Assemble the MicroElute HiBind DNA Column into a 2 ml collection tube(supplied). Spin for 2 min at maximum speed ( 13,000 x g) to dry the MicroElute HiBind DNA column Place the MicroElute HiBind DNA Column in a clean 1.5 ml microcentrifuge tube(not supplied) and add μl of Elution Buffer. Incubate at room temperature for 1-2 minutes. Centrifuge for 1 min at 13, 000 x g to elute DNA.

15 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 ( ). Possible Problems and Suggestions Low DNA Yields Not enough CP Buffer added to sample Add more CP Buffer as indicated. For DNA fragments < 200bp in size, add up to 6 x volumes of CP Buffer. ph of the sample mixture is too high. Add μl of Sodium Acetate ph 5.2 to the sample and mix. Clogged Column in Gel Extraction Incompletely dissolved Gel. Increase incubation time. Increase Binding Buffer Volume. No DNA Eluted SPW or DNA Wash Buffer not diluted with ethanol. Prepare SPW or DNA Wash Buffer Concentrate as instructed. Optical densities do not agree with DNA yield on agarose gel. Trace contaminants eluted from column increase A 260. Wash column as instructed. Alternatively, rely on agarose gel/ethidium bromide electrophoresis for quantification. DNA sample floats out of well while loading agarose gel. Ethanol not removed completely from column following wash steps. Centrifuge column as instructed to dry before proceeding to elution. 15

16 Ordering Information The following components are available for purchase separately. (Call Toll Free Number ) Buffer (Size) CP Buffer (200 ml) Binding Buffer (200 ml) DP Buffer (100 ml) Elution Buffer (100 ml) DNA Wash Buffer (100ml) MicroElute DNA Columns (50) SPW Wash Buffer (25ml) 2ml capless collection tubes 1.5ml DNase/RNase Free Centrifuge Tubes Part Number PDR042 PDR040 PDR047 PDR048 PS010 MEDNACOL-01 PDR045 SS SS Hibind, E.Z.N.A and MicroElute are registered trademarks of Omega Bio-tek, Inc. Qiagen, QIAvac and Vacman are all trademarks of their respected companies. PCR is a patented process of Hoffman-La Roche. Use of PCR process requires a license. 16