UltraClean PCR Clean-Up Kit Sample - MO BIO Laboratories

UltraClean ®PCR Clean-Up Kit Sample(Catalog No. 12500-S)Information for Ordering ProductCatalog No. Quantity12500-50 50 Preps12500-100 100 Preps12500-250 250 PrepsInstruction ManualNew protocol instruction: Shake the SpinBind Solution before using to ensure consistent results.Please recycleVersion: 10282009Technical information: Toll free 1-800-606-6246, or 1-760-929-9911 Email: technical@mobio.com Website: www.mobio.com1

IntroductionThe UltraClean ® PCR Clean-Up Kit is designed to purify PCR products directly from a PCR or enzymereaction in just 3 minutes without running an agarose gel. If you sequence your PCR reactions or haveapplications where efficient removal of PCR primers is critical, this kit is your solution. All reagents areoptimized to remove primers, dNTPs and reaction components while purifying PCR reaction products inthe size range of 60 bp to 10 kb.Protocol OverviewWith the addition of binding buffer, a novel silica spin filter is used to selectively bind the PCR or reactionproduct. Unwanted reaction components are passed through the filter by centrifugation. The desired product isthen washed and recovered from the spin filter in certified DNA-free Tris buffer. The resulting DNA can be usedfor any downstream application.SpecificationsDNA Size range: 60 bp - 10 kbBinding capacity of Spin Filter: 20 gFinal volume of DNA: 50 lRecovery rates: 80-100%High Throughput OptionsMO BIO offers a vacuum based protocol for faster processing without centrifugation for the DNA bindingand column washing steps for Spin Filters. The MO BIO PowerVac Manifold allows for processing ofup to 20 spin filter preps at a time using the PowerVac Mini Spin Filter Adapters. For additional highthroughput options MO BIO offers the UltraClean ® -htp 96 Well PCR Clean-Up Kit for processing up to 2x 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 PlateAdapter Set (MO BIO Catalog# 11996 & 11999, respectively.)This kit is for research purposes only. Not for diagnostic use.Other Related Products Catalog No. QuantityUltraClean ® -htp 96 Well PCR Clean-Up Kit 12596-412596-124 x 96 preps12 x 96 prepsUltraClean ® Agarose, Molecular Biology Grade 15003-5015003-10015003-50015003-100050 g100 g500 g1 kgUltraClean ® Lab Cleaner 12095-25012095-50012095-1000250 ml bottle500 ml bottle1 L bottlePowerVac Manifold 11991 1 manifoldPowerVac Mini System 11992 1 unit + 20adaptersPowerVac Mini Spin Filter Adapters 11992-1011992-2010 adapters20 adaptersTechnical information: Toll free 1-800-606-6246, or 1-760-929-9911 Email: technical@mobio.com Website: www.mobio.com3

Technical information: Toll free 1-800-606-6246, or 1-760-929-9911 Email: technical@mobio.com Website: www.mobio.com4

Experienced User ProtocolPlease wear gloves at all times1. 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 10-30 seconds at a minimum 10,000 x g (approximately 13,000 rpm) in a tabletopmicrocentrifuge.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 10-30 seconds at a minimum 10,000 x g.9. Remove Spin Filter basket and discard liquid flow through by decanting then replace basket back intothe same tube.10. Centrifuge 30-60 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 ofthe 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 30-60 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 mlCollection Tube. The DNA will be free of all reaction components such as primers or linkers,enzyme, salt, and dNTP’s. Store DNA at -20C. DNA is now ready to use.Thank you for choosing the UltraClean ® PCR Clean-Up Kit Sample.Technical information: Toll free 1-800-606-6246, or 1-760-929-9911 Email: technical@mobio.com Website: www.mobio.com6

Detailed Protocol (Describes what is happening at each step)Please wear gloves at all times1. 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 arecreating a pH buffered high salt condition. pH is critical after the addition of SpinBind. pH above 8 results in lowDNA recovery. pH below 5, results in primers co-purifying with your sample DNA. Optimal pH range is 6-7.5 forhigh 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 atthis step. If you used a wax overlay, try to avoid carrying over any during this step.4. Centrifuge 10-30 seconds at a minimum 10,000 x g (approximately 13,000 rpm) in a tabletopmicrocentrifuge.What’s happening: DNA in the size range from 60 bp to 10 kb will bind to the white silica spin filter membraneat the bottom of the silica spin filter unit. The liquid that passes through the membrane will contain unwantedcomponents of the PCR reaction such as: PCR primers, dNTPs, enzyme, and buffer constituents. The desiredPCR 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 labsafety office for proper disposal and handling.6. Replace the Spin Filter basket in the same tube.7. Add 300l SpinClean Buffer to the Spin Filter.What’s happening: SpinClean is a wash solution. It will remove any traces of unwanted contaminants whileallowing the desired PCR product DNA to stay bound to the silica spin filter membrane. SpinClean is anethanol based wash solution. It contains less than 80% ethanol. This solution is flammable so use caution nearflames.8. Centrifuge 10-30 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 intothe same tube.10. Centrifuge 30-60 seconds at minimum 10,000 x g.Technical information: Toll free 1-800-606-6246, or 1-760-929-9911 Email: technical@mobio.com Website: www.mobio.com7

What’s happening: This step will remove any last traces of SpinClean. The ethanol in the SpinClean can have anegative 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 ofthe 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 surethe entire membrane is wetted. This will result in more efficient release of the desired DNA.13. Centrifuge 30-60 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 flowsthrough the membrane, and into the collection tube. The DNA is released because it can only bind to the silicaspin 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 -20C.What’s happening: Purified DNA is now in the collection tube. The DNA is UltraClean and free of all reactioncomponents such as primers or linkers, enzyme, salt, and dNTP’s. The DNA is now ready to use for anyapplication. 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 Sample.Technical information: Toll free 1-800-606-6246, or 1-760-929-9911 Email: technical@mobio.com Website: www.mobio.com8

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 -80C). Elution Buffer is 10 mM Tris and contains noEDTA. To concentrate the DNA see the Hints & Troubleshooting Guide.Thank you for choosing the UltraClean ® PCR Clean-Up Kit Sample.Technical information: Toll free 1-800-606-6246, or 1-760-929-9911 Email: technical@mobio.com Website: www.mobio.com10

Hints and Troubleshooting GuideConcentrating the DNAYour final volume will be 50 l. If this is too dilute for your purposes, add 2 l of 5 M NaCl and mix. Thenadd 100 l of 100% cold ethanol. Mix. Centrifuge at 10,000 x g for 5 minutes. Decant all liquid. Dryresidual ethanol in a speed vac or desiccator or ambient air. Resuspend precipitated DNA in desiredvolume.DNA Floats Out of Well When Loaded on a GelResidual SpinClean in the final sample. Prevent this by being careful in step 11 not to transfer liquid ontothe bottom of the spin filter basket. Ethanol precipitate to remove residues of SpinClean. (See procedurefor “Concentrating the DNA” above.)Low RecoveryLow recoveries can be due to not mixing SpinBind well with your sample in step 2. Incomplete removal ofSpinClean can also reduce yields. Make sure your centrifuge is spinning at 10,000 x g minimum.Enzyme Reactions InhibitedIf you chose to elute in a buffer containing EDTA, you may see inhibition of subsequent enzymaticreactions. Re-purify the sample with this kit and use the Tris solution Elution Buffer provided for theelution step.Cleaning of the PowerVac Mini Spin Filter AdaptersIt is recommended to rinse the PowerVac Mini Spin Filter Adapters promptly after use to avoid salt buildup. 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 laboratorydetergent 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 thePowerVac Manifold. Bleach should never be mixed with solutions containing guanidine andshould not be used to clean the PowerVac Manifold. For more information on cleaning thePowerVac Manifold, please refer to the PowerVac Manifold manual.Technical information: Toll free 1-800-606-6246, or 1-760-929-9911 Email: technical@mobio.com Website: www.mobio.com11

Contact InformationTechnical Support:Phone MO BIO Laboratories, Inc. Toll Free 800-606-6246, or 760-929-9911Email: technical@mobio.comFax: 760-929-0109Mail: MO BIO Laboratories, Inc, 2746 Loker Ave West, Carlsbad, CA 92010Ordering Information:Direct: Phone MO BIO Laboratories, Inc. Toll Free 800-606-6246, or 760-929-9911Email: orders@mobio.comFax: 760-929-0109Mail: MO BIO Laboratories, Inc, 2746 Loker Ave West, Carlsbad, CA 92010For the distributor nearest you, visit our web site at www.mobio.com/distributorsTechnical information: Toll free 1-800-606-6246, or 1-760-929-9911 Email: technical@mobio.com Website: www.mobio.com12

Other Quality Products Available from MO BIO Laboratories, Inc.For more product and detailed information go to www.mobio.com/catalog-request to request a catalog.Genomic DNA Isolation…Continued Catalog No. QuantityUltraClean® Forensic DNA Isolation 14000-10 10 isolationsKit14000-20 20 isolationsPowerWater® DNA Isolation Kit14900-50-NF14900-50-2214900-50-45RapidWater DNA Isolation KitUltraClean® Water DNA Isolation Kit(0.45µm filters)UltraClean® Water DNA Isolation Kit(0.22 µm filters)UltraClean® Water DNA Isolation Kit(No filters)14900-100-NF14900-100-2214900-100-4514810-50-NF14810-50-2214810-50-4514810-100-NF14810-100-2214810-100-4514800-1014800-2514880-1014880-2514800-10-NF14800-25-NF50 preps(No filters)(0.22 μm)(0.45 μm)100 preps(No filters)(0.22 μm)(0.45 μm)50 preps(No filters)(0.22 μm)(0.45 μm)100 preps(No filters)(0.22 μm)(0.45 μm)10 preps25 preps10 preps25 preps10 preps25 prepsMicrobiological Culture Media Catalog No. QuantityTB DRY® Powder Growth Media 12105-05 500 g12105-1 1 kg12105-5 5 kgLB Broth Powder Growth Media, pH712106-0512106-112106-5LB Agar Powder Growth Media, pH 7 12107-0512107-112107-5LB Broth (Lennox) Powder GrowthMedia, pH 7LB Agar (Lennox) Powder GrowthMedia, pH 7Soybean-Casein Digest Medium(TSB), USPSoybean-Casein Digest AgarMedium (TSA), USP12108-0512108-112108-512109-0512109-112109-512114-0512114-112114-512115-0512115-112115-5Yeast Extract 12110-0512110-112110-5Tryptone 12111-0512111-112111-5Agar, Bacteriological Grade 12112-0512112-112112-5Other Reagents and Lab500 g1 kg5 kg500 g1 kg5 kg500 g1 kg5 kg500 g1 kg5 kg500 g1 kg5 kg500 g1 kg5 kg500 g1 kg5 kg500 g1 kg5 kg500 g1 kg5 kgOther Reagents and LabAccessories…Continued Catalog No. Quantity100 bp DNA Ladder 17100-40 40 µg1 kb DNA Ladder 17200-100 100 µgUltraClean® Agarose, MolecularBiology Grade15003-5015003-10015003-50015003-1000UltraClean® MS-8 Agarose 15515-5015515-10015515-500UltraClean® Forensic Agarose 15505-5015505-10015505-500UltraClean® Low Melt Agarose 15005-5015005-10015005-500UltraClean® Low Melt Sieve Agarose 15004-5015004-10015004-500Ethidium Bromide Solution 15006-115006-10Ethidium Bromide Destaining TeaBagsBromophenol Blue Gel LoadingBufferBromophenol Blue/Xylene CyanolGel Loading BufferTechnical information: Toll free 1-800-606-6246, or 1-760-929-9911 Email: technical@mobio.com Website: www.mobio.com1450 g100 g500 g1 kg50 g100 g500 g50 g100 g500 g50 g100 g500 g50 g100 g500 g1 ml10 ml15007-25 25 bags15008-115008-515009-115009-5TAE Buffer, 50X (Tris-acetate-EDTA) 15001-10015001-50015001-1000TBE Buffer, 10X (Tris-borate-EDTA) 15002-10015002-50015002-1000RNase-Free Gloves1555-XS1555-S1555-M1555-LUltraClean® Lab Cleaner 12095-250OmniTaq DNA PolymeraseEnzymeOmniTaq DNA Polymerase 2xMaster MixAccessories Catalog No. QuantityOmni KlenTaq DNA PolymeraseEnzyme20 bp DNA Ladder 17020-40 40 µg Omni KlenTaq DNA Polymerase 2xMaster Mix12095-5001 ml5 x 1 ml1 ml5 x 1 ml100 ml500 ml1 liter100 ml500 ml1 literbag of 100bag of 100bag of 100bag of 100250 mlsqueeze bottle500 ml spraybottle1 liter bottle12095-10001224-250 250 reactions(10 U/µl)1226-250 250 reactions(5 x 1.25ml/tube)1225-250 250 reactions(25 U/µl)1227-250 250 reactions(5 x 1.25ml/tube)

Other Quality Products Available from MO BIO Laboratories, Inc.For more product and detailed information go to www.mobio.com/catalog-request to request a catalog.Other Reagents and LabAccessories…Continued Catalog No. QuantityDNase (RNase-Free) 15600-5 5 mg15601-100 2500 unitsProteinase K 1223-1001222-2Ribonuclease A (25 mg/ml) 1202-11202-5PCR Water 17000-117000-517000-1017000-11Molecular Biology Grade Water 17012-20017012-5200DEPC Treated Water 17011-20017011-5200100 mg2 ml (20mg/ml)1 ml5 ml1 ml5 x 1 ml10 x 1 ml10 ml bottle200 ml5 x 200 ml200 ml5 x 200 mlInstrumentation andAccessories… Continued Catalog No. QuantityGlass 50 ml Bead Tubes, 0.1 mm 13145-10 10 tubes13145-50 50 tubes13145-100 100 tubes13145-500 500 tubesGlass 15 ml Bead Tubes, 1.0 mm 13136-50 50 tubesCeramic 15 ml Bead Tubes, 1.4 mm 13137-50 50 tubesCeramic 50 ml Bead Tubes, 1.4 mm 13147-1013147-5010 tubes50 tubesMetal 50 ml Bead Tubes, 2.38 mm 13149-10 10 tubes13149-50 50 tubesPowerMix 15 ml Bead Tubes 13138-50 50 tubesEndotoxin-Free Water 17013-1017013-5017013-10017013-50010 ml50 ml100 ml500 mlPowerMix 50 ml Bead Tubes 13148-1013148-502 ml Collection Tubes 1200-100-T1200-150-T1200-250-T10 tubes50 tubes100 tubes150 tubes250 tubesInstrumentation and Accessories Catalog No. QuantityBagMixer® 400 VW 23112 1 unit 2 ml Screw Cap Tubes 12800-200-E 200 tubes &capsBagFilter® 400 P 23113-500 Box of 500 15 ml Collection Tubes 12700-T 25 tubesBagPage® 400 23114-500 Box of 500 50 ml Centrifuge Tubes 12600-T 25 tubesPrecellys® 24 Homogenizer, 120V 13112 1 unit Spin Filters (in 1.9 ml tubes) 1200-50-SF1200-100-SF1200-250-SFCeramic Bead Tubes, 1.4 mm 13113-50 50 bead tubes Endotoxin-Free Centrifuge Tubes 12617-10012618-5012619-2550 filters100 filters250 filters100 each/2 mltubes50 each/15 mltubes25 each/50 mltubesCeramic Bead Tubes, 2.8 mm 13114-50 50 bead tubes 15 ml Midi Spin Filters 12700-SF 25 spin filtersGlass Bead Tubes, 0.5 mm 13116-50 50 bead tubes Vortex-Genie® 2 Vortex (120V) 13111-V 1 unitGlass Bead Tubes, 0.1 mm 13118-50 50 bead tubes Vortex-Genie® 2 Vortex (220V) 13111-V-220 1 unitMetal Bead Tubes, 2.38 mm 13117-50 50 bead tubes Vortex Adapter, holds 12 (1.5-2.0 ml) 13000-V1 1 unittubes2.0 ml Tough Tubes with Cap 13119-500 500Vortex Adapter, holds 6 (5 ml) tubes 13000-V1-5 1 unit13119-1000 1000Carbide Bead Tubes, 0.25 mm 13121-50 50 x 0.5 mlVortex Adapter, holds 4 (15 ml) tubes 13000-V1-15 1 unittubesGarnet Bead Tubes, 0.15 mm 13122-50 50 x 0.5 mlVortex Adapter, holds 2 (50 ml) tubes 13000-V1-50 1 unittubesGarnet Bead Tubes, 0.70 mm 13123-50 50 x 2 mlVortex Adapter, holds 24 (1.5-2.0 ml) 13000-V1-24 1 unittubestubesGarnet + ¼ Ceramic 15 ml Bead 13134-50 50 tubes Power Supply w/Timer, (120V) 16023 1 unitTubes, 0.70 mmGarnet + ¼ Ceramic 50 ml BeadTubes, 0.70 mm `Power Supply w/Timer, (220V) 16023-220 1 unit13144-1013144-5013144-10013144-50010 tubes50 tubes100 tubes500 tubesGlass 15 ml Bead Tubes, 0.1 mm 13135-50 50 tubes Polycarbonate Single-sided Comb 160051600616007160081 mm x 3 well1 mm x 8 well1 mm x 10 well1 mm x 12 wellTechnical information: Toll free 1-800-606-6246, or 1-760-929-9911 Email: technical@mobio.com Website: www.mobio.com15

Other Quality Products Available from MO BIO Laboratories, Inc.For more product and detailed information go to www.mobio.com/catalog-request to request a catalog.Instrumentation andAccessories… Continued Catalog No. QuantityPolycarbonate Dual-sided Comb 160131 mm x 8well/16 well160141 mm x 10well/14 well160152 mm x 8well/16 well160162 mm x 10well/14 wellTeflon Single-sided Comb 160091 mm x 3 well160101 mm x 8 well160111 mm x 10 well160121 mm x 12 wellTeflon Dual-sided Comb 160171 mm x 8well/16 well160181 mm x 10well/14 well160192 mm x 8well/16 well160202 mm x 10well/14 wellInstrumentation andAccessories… Continued Catalog No. Quantity96 Well Plate Shaker (220V) 11996-220 1 unitPlate Adapter Set 11999 1 setTube Adapter Set 11995 1 setMini Horizontal Gel System 16001 1 each Vacuum Pump (120V) 11998 1 unitMini Horizontal Gel Caster, 3 place 16003 1 each Vacuum Pump (220V) 11998-220 1 unitMini Horizontal Gel Tray 16004 1 each UltraVac Manifold 11997 1 unit96 Well Plate Shaker (120V) 11996 1 unitTechnical information: Toll free 1-800-606-6246, or 1-760-929-9911 Email: technical@mobio.com Website: www.mobio.com16