UltraClean PCR Clean-Up Kit

Transcription

1 UltraClean PCR Clean-Up Kit Catalog No. Quantity Preps Preps Preps Instruction Manual Please recycle Version:

2 Table of Contents Introduction... 3 Protocol Overview... 3 Specifications... 3 Flow Chart... 4 Equipment Required... 5 Kit Contents & Storage... 5 Precautions & Warnings... 5 Protocols: Experienced User Protocol... 6 Detailed Protocol (Describes what is happening at each step)... 7 Vacuum Manifold Protocol... 9 Hints & Troubleshooting Guide Contact Information Products recommended for you

3 Introduction The UltraClean PCR Clean-Up Kit is designed to purify PCR products directly from a PCR or enzyme reaction in just 3 minutes without running an agarose gel. If you sequence your PCR reactions or have applications where efficient removal of PCR primers is critical, this kit is your solution. All reagents are optimized to remove primers, dntps and reaction components while purifying PCR reaction products in the size range of 60 bp to 10 kb. Protocol Overview With the addition of binding buffer, a novel silica spin filter is used to selectively bind the PCR or reaction product. Unwanted reaction components are passed through the filter by centrifugation. The desired product is then washed and recovered from the spin filter in certified DNA-free Tris buffer. The resulting DNA can be used for any downstream application. Specifications DNA Size range: 60 bp - 10 kb Binding capacity of Spin Filter: 20 g Final volume of DNA: 50 l Recovery rates: % High Throughput Options MO BIO offers a vacuum based protocol for faster processing without centrifugation for the DNA binding and column washing steps for Spin Filters. The MO BIO PowerVac Manifold allows for processing of up to 20 spin filter preps at a time using the PowerVac Mini Spin Filter Adapters. For additional high throughput options MO BIO offers the UltraClean -htp 96 Well PCR Clean-Up Kit for processing up to 2 x 96 samples using a centrifuge capable of spinning two 96 Well Blocks stacked (13 cm x 8 cm x 5.5 cm) at 2500 x g. For 96 well homogenization of soil, MO BIO offers the 96 Well Plate Shaker and Plate Adapter Set (MO BIO Catalog# & 11990, respectively.) This kit is for research purposes only. Not for diagnostic use. Other Related Products Catalog No. Quantity UltraClean -htp 96 Well PCR Clean-Up Kit x 96 preps 12 x 96 preps UltraClean Agarose, Molecular Biology Grade g 100 g UltraClean Lab Cleaner ml bottle 500 ml bottle 1 L bottle PowerVac Manifold manifold PowerVac Mini System unit + 20 adapters PowerVac Mini Spin Filter Adapters adapters 20 adapters 3

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5 Equipment Required Microcentrifuge (10,000 x g) Pipettor (volumes required 50 l l) Vortex-Genie 2 Vortex (MO BIO Catalog# V or V-220) Reagents Required but not Included 100% ethanol (for the PowerVac Manifold protocol only) Kit Contents Kit Catalog # Kit Catalog # Kit Catalog # Component Catalog # Amount Catalog # Amount Catalog # Amount SpinBind ml ml ml SpinClean Buffer ml ml x 30 ml Elution Buffer ml ml ml Spin Filters SF SF SF 250 (Units in 2 ml Tubes) 2 ml Collection Tubes T T T 250 Kit Storage Kit reagents and components should be stored at room temperature (15-30 C). Precautions Please wear gloves when using this product. Avoid all skin contact with kit reagents. In case of contact, wash thoroughly with water. Do not ingest. See Material Safety Data Sheets for emergency procedures in case of accidental ingestion or contact. All MSDS information is available upon request ( ) or at Reagents labeled flammable should be kept away from open flames and sparks. WARNING: SpinClean Buffer contains ethanol. It is flammable. Do not use bleach to clean the inside of the PowerVac Manifold or to rinse the PowerVac Mini Spin Filter Adapters when attached to the manifold. IMPORTANT NOTE FOR USE: Shake to mix the SpinBind before use. 5

6 Experienced User Protocol Please wear gloves at all times 1. Shake to mix the SpinBind before use. Add 5 volumes of SpinBind to your PCR reaction. Example: add 500 l to a 100 l PCR reaction. 2. Mix well by pipetting. If an oil overlay was used, you will now have two layers. The top layer is oil. 3. Transfer PCR/SpinBind mixture to a Spin Filter unit, while avoiding the transfer of oil. 4. Centrifuge seconds at a minimum 10,000 x g (approximately 13,000 rpm) in a tabletop microcentrifuge. 5. Remove the Spin Filter basket and discard the liquid flow-through from the tube by decanting. 6. Replace the Spin Filter basket in the same tube. 7. Add 300 l SpinClean Buffer to the Spin Filter. 8. Centrifuge seconds at a minimum 10,000 x g. 9. Remove Spin Filter basket and discard liquid flow through by decanting then replace basket back into the same tube. 10. Centrifuge seconds at minimum 10,000 x g. 11. Transfer Spin Filter to a clean 2 ml Collection Tube (provided). 12. Add 50 l of Elution Buffer (10 mm Tris) solution provided or sterile water directly onto the center of the white Spin Filter membrane. The choice of using Tris or water at this point will not affect yield. DNA is more stable for storage in Tris. 13. Centrifuge seconds at a minimum 10,000 x g. 14. Discard Spin Filter basket from the inside of the Spin Filter unit. Purified DNA is now in the 2 ml Collection Tube. The DNA will be free of all reaction components such as primers or linkers, enzyme, salt, and dntp s. Store DNA at -20 C. DNA is now ready to use. Thank you for choosing the UltraClean PCR Clean-Up Kit. 6

7 Detailed Protocol (Describes what is happening at each step) Please wear gloves at all times 1. Shake to mix the SpinBind before use. Add 5 volumes of SpinBind to the PCR reaction. Example: add 500 l to a 100 l PCR reaction. What s happening: SpinBind is a buffered salt solution. By mixing it with your PCR reaction product, you are creating a ph buffered high salt condition. ph is critical after the addition of SpinBind. ph above 8 results in low DNA recovery. ph below 5, results in primers co-purifying with your sample DNA. Optimal ph range is for high recovery and total primer removal. 2. Mix well by pipetting. If an oil overlay was used, you will now have two layers. The top layer is oil. What s happening: Mixing well creates a homogeneous salt concentration throughout the sample tube. 3. Transfer PCR/SpinBind mixture to a Spin Filter unit, while avoiding the transfer of oil. What s happening: Although the oil does not affect DNA binding, it can be very messy if you do not remove it at this step. If you used a wax overlay, try to avoid carrying over any during this step. 4. Centrifuge seconds at a minimum 10,000 x g (approximately 13,000 rpm) in a tabletop microcentrifuge. What s happening: DNA in the size range from 60 bp to 10 kb will bind to the white silica spin filter membrane at the bottom of the silica spin filter unit. The liquid that passes through the membrane will contain unwanted components of the PCR reaction such as: PCR primers, dntps, enzyme, and buffer constituents. The desired PCR product DNA will bind to silica under high salt conditions. 5. Remove the Spin Filter basket and discard the liquid flow-through from the tube by decanting. What s happening: This is flow through waste. This contains Guanidine HCl so be sure to check with your lab safety office for proper disposal and handling. 6. Replace the Spin Filter basket in the same tube. 7. Add 300 l SpinClean Buffer to the Spin Filter. What s happening: SpinClean is a wash solution. It will remove any traces of unwanted contaminants while allowing the desired PCR product DNA to stay bound to the silica spin filter membrane. SpinClean is an ethanol based wash solution. It contains less than 80% ethanol. This solution is flammable so use caution near flames. 8. Centrifuge seconds at a minimum 10,000 x g. What s happening: As the SpinClean passes through the spin filter membrane, it cleans the PCR product DNA. 9. Remove Spin Filter basket and discard liquid flow through by decanting then replace basket back into the same tube. 7

8 10. Centrifuge seconds at minimum 10,000 x g. What s happening: This step will remove any last traces of SpinClean. The ethanol in the SpinClean can have a negative affect on the DNA purity. 11. Transfer Spin Filter to a clean 2 ml Collection Tube (provided). 12. Add 50 l of Elution Buffer (10mM Tris) solution provided or sterile water directly onto the center of the white Spin Filter membrane. The choice of using Tris or water at this point will not affect yield. DNA is more stable for storage in Tris. What s happening: Placing the Elution Buffer in the center of the small white silica membrane will make sure the entire membrane is wetted. This will result in more efficient release of the desired DNA. 13. Centrifuge seconds at a minimum 10,000 x g. What s happening: As the Elution Buffer passes through the silica membrane, DNA is released, and it flows through the membrane, and into the collection tube. The DNA is released because it can only bind to the silica spin filter membrane in the presence of salt. Elution Buffer is 10mM Tris ph. 8 and does not contain salt. 14. Remove Spin Filter basket from the 2 ml Collection Tube. Seal tube and store DNA at -20 C. What s happening: Purified DNA is now in the collection tube. The DNA is UltraClean and free of all reaction components such as primers or linkers, enzyme, salt, and dntp s. The DNA is now ready to use for any application. The DNA is in a 50 l volume. To concentrate it, see the Hints and Troubleshooting Guide. Thank you for choosing the UltraClean PCR Clean-Up Kit. 8

9 Vacuum Protocol using the PowerVac Manifold Please wear gloves at all times For each sample lysate, use one Spin Filter column. Keep the Spin Filter in the attached 2 ml Collection Tube and continue using the Collection Tube as a Spin Filter holder until needed for the Vacuum Manifold Protocol. Label each Collection Tube top and Spin Filter column to maintain sample identity. You will need to provide 100% ethanol for step 4 of this protocol 1. For each prep, attach one aluminum PowerVac Mini Spin Filter Adapter (MO BIO Catalog# or ) into the Luer-Lok fitting of one port in the manifold. Gently press a Spin Filter column into the PowerVac Mini Spin Filter Adapter until snugly in place. Ensure that all unused ports of the vacuum manifold are closed. Note: Aluminum PowerVac Mini Spin Filter Adapters are reusable. 2. Transfer up to 650 l of prepared sample lysate (from step 2) to the Spin Filter column. 3. Turn on the vacuum source and open the stopcock of the port. Hold the tube in place when opening the stopcock to keep the spin filter steady. Allow the lysate to pass through the Spin Filter column. After the lysate has passed through the column completely, if you have more sample, load again with the next 650 l until all of the lysate has been loaded onto the Spin Filter column. Close the oneway Luer-Lok stopcock of that port. Note: If Spin Filter columns are filtering slowly, close the ports to samples that have completed filtering to increase the pressure to the other columns. 4. Load 800 l of 100% ethanol into the Spin Filter so that it completely fills the column. Open the stopcock while holding the column steady. Allow the ethanol to pass through the column completely. Close the stopcock. 5. Add 300 l of SpinClean Buffer to each Spin Filter. Open the Luer-Lok stopcock and apply a vacuum until SpinClean Buffer has passed through the Spin Filter completely. Continue to pull a vacuum for another minute to dry the membrane. Close each port. 6. Turn off the vacuum source and open an unused port to vent the manifold. If all 20 ports are in use, break the vacuum at the source. Make certain that all vacuum pressure is released before performing the next step. It is important to turn off the vacuum at the source to prevent backflow into the columns. 7. Remove the Spin Filter column and place in the original labeled 2 ml Collection Tube. Place into the centrifuge and spin at 13,000 g for 1 minute to completely dry the membrane. 8. Transfer the Spin Filter column to a new 2 ml Collection Tube and add 50 l of Elution Buffer to the center of the white filter membrane. 9. Centrifuge at room temperature for 30 seconds at 10,000 x g. 9

10 10. Discard the Spin Filter column. The DNA in the tube is now ready for any downstream application. No further steps are required. We recommend storing DNA frozen (-20 to -80 C). Elution Buffer is 10 mm Tris and contains no EDTA. To concentrate the DNA see the Hints & Troubleshooting Guide. Thank you for choosing the UltraClean PCR Clean-Up Kit. 10

11 Hints and Troubleshooting Guide Concentrating the DNA Your final volume will be 50 l. If this is too dilute for your purposes, add 2 l of 5 M NaCl and mix. Then add 100 l of 100% cold ethanol. Mix. Centrifuge at 10,000 x g for 5 minutes. Decant all liquid. Dry residual ethanol in a speed vac or desiccator or ambient air. Resuspend precipitated DNA in desired volume. DNA Floats Out of Well When Loaded on a Gel Residual SpinClean in the final sample. Prevent this by being careful in step 11 not to transfer liquid onto the bottom of the spin filter basket. Ethanol precipitate to remove residues of SpinClean. (See procedure for Concentrating the DNA above.) Low Recovery Low recoveries can be due to not mixing SpinBind well with your sample in step 2. Incomplete removal of SpinClean can also reduce yields. Make sure your centrifuge is spinning at 10,000 x g minimum. Enzyme Reactions Inhibited If you chose to elute in a buffer containing EDTA, you may see inhibition of subsequent enzymatic reactions. Re-purify the sample with this kit and use the Tris solution Elution Buffer provided for the elution step. Cleaning of the PowerVac Mini Spin Filter Adapters It is recommended to rinse the PowerVac Mini Spin Filter Adapters promptly after use to avoid salt build up. To clean the PowerVac Mini Spin Filter Adapters, rinse each adapter with DI water followed by 70% ethanol and flush into the manifold base. Alternatively, remove the adapters and wash in laboratory detergent and DI water. PowerVac Mini Spin Filter Adapters may be autoclaved. Do not use bleach to clean the PowerVac Mini Spin Filter Adapters while attached to the PowerVac Manifold. Bleach should never be mixed with solutions containing guanidine and should not be used to clean the PowerVac Manifold. For more information on cleaning the PowerVac Manifold, please refer to the PowerVac Manifold manual. 11

12 Contact Information Technical Support: Phone MO BIO Laboratories, Inc. Toll Free , or Fax: Mail: MO BIO Laboratories, Inc, 2746 Loker Ave West, Carlsbad, CA Ordering Information: Direct: Phone MO BIO Laboratories, Inc. Toll Free , or Fax: Mail: MO BIO Laboratories, Inc, 2746 Loker Ave West, Carlsbad, CA For the distributor nearest you, visit our web site at 12

13 Products recommended for you For a complete list of products available from MO BIO Laboratories, Inc., visit Description Catalog No. Quantity x 96 preps UltraClean -htp 96 Well PCR Clean-Up Kit x 96 preps UltraClean GelSpin DNA Extraction Kit preps 100 preps 250 preps UltraClean 15 DNA Purification Kit preps PowerClean DNA Clean-Up Kit preps Dye Dots plates plates 13