On-Spin Column DNase I Kit (RNase-Free)

Transcription

1 On-Spin Column DNase I (RNase-Free) Catalog No. Quantity Instruction Manual F Please recycle Version:

2 Introduction Use this product to remove contaminating genomic DNA from silica membrane Spin Filters prior to RNA elution. The On-Spin Column DNase I is a simple and effective method for the removal of DNA during the RNA isolation process. The MO BIO Laboratories On-Spin Column DNase I is compatible with all silica membrane spin column based RNA isolation kits. Note: This kit can also be used for purified RNA already in solution. *This kit is for research purposes only. Not for diagnostic use. Equipment Required Microcentrifuge (13,000 x g) Pipet (volumes required 1.5 l l) Vortex Contents Components Amount Solution D1 (DNase I Buffer) 2.5 ml Solution D2 (DNase Wash Buffer) 25 ml Solution D3 (Wash Buffer II) 2 x 28 ml DNase I (RNase-Free); Lyophilized powder 1500 units Sterile Water (DNase I Resuspension Water) 1 ml 2 ml Collection Tubes 50 Storage Remove DNase I (RNase-Free) lyophilized powder and store at 4ºC. The lyophilized powder will be resuspended to make a DNase I stock enzyme solution. After resuspension, store DNase I stock enzyme at -20 C (DNase I is sensitive to physical denaturation. Do not vortex the resuspended DNase I stock enzyme). Store all other components at room temperature (15-30ºC). Precautions Please wear gloves when using this product. Avoid all skin contact with reagents in this kit. 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 on our web site at Reagents labeled flammable should be kept away from open flames and fire. Warning: Solution D3 contains ethanol. It is flammable. To prevent fire danger, avoid open flames and heat sources. 2

3 Important Notes Before Starting: DNase I Stock Enzyme and DNase I Solution Preparation and Storage A. Prepare DNase I stock enzyme by adding 300 µl of Sterile Water (provided) to the DNase I (RNase- Free) lyophilized powder and mix gently. Aliquot the DNase I stock enzyme in 50 µl portions and store at -20 C for long term storage. Note: The DNase I stock enzyme can be freeze/thawed up to three times without loss of activity. B. Prepare the DNase I Solution, by thawing the volume of DNase I stock enzyme needed according to the number of samples. Per prep, combine 5 µl of DNase I stock enzyme with 45 µl of Solution D1. Example: Number DNase I stock Solution D1 of preps enzyme 1 5 µl 45 µl 2 10 µl 90 µl µl 450 µl 3

4 Protocol On-Spin Column DNase I Treatment: 1. After loading the RNA lysate onto a Spin Filter, wash the column using the wash buffer provided in your kit. If you are using a MO BIO RNA Isolation, the table below lists the Wash Buffer name and volume used depending on the kit you have. Centrifuge the wash buffer through the column for 2 minutes at 10,000 x g to thoroughly remove all traces of wash buffer from the Spin Filter membrane. Note: If using another manufacturer s silica membrane spin filter or spin column RNA isolation kit, follow the protocol up to the point where RNA is washed just prior to the final RNA release or elution step. MO BIO RNA Isolation Catalog # Wash Buffer Name Buffer Volume UltraClean Tissue & Cells Total RNA Isolation Solution TR3 500 µl UltraClean Microbial RNA Isolation Solution MR5 300 µl UltraClean Plant RNA Isolation Solution PMR5 500 µl BiO stic Blood Total RNA Isolation Solution BR4 400 µl BiO stic Stabilized Blood RNA Isolation Solution PX5 400 µl 2. Transfer the Spin Filter to a new 2 ml Collection Tube (provided). To the center of the Spin Filter, add 50 µl of DNase I Solution (a mixture of 5 μl of DNase I stock enzyme and 45 µl of Solution D1, see DNase I Solution Preparation and Storage in the Important Notes Before Starting section on the previous page). 3. Incubate at room temperature for 15 minutes. 4. To the Spin Filter column add 400 µl Solution D2 and centrifuge at 10,000 x g for 1 minute. Discard the flow through and place the Spin Filter back into the 2 ml Collection Tube. 5. Add 500 µl of Solution D3 to the Spin Filter. Centrifuge at 10,000 x g for 1 minute. Discard the flow through and place the Spin Filter back into the 2 ml Collection Tube. 6. Repeat the wash by adding another 500 µl of Solution D3 to the Spin Filter. Centrifuge at 10,000 x g for 1 minute. Discard the flow through and place the Spin Filter back into the 2 ml Collection Tube. Centrifuge the Spin Filter for 2 minutes at 13,000 x g to completely dry the membrane. Continue with the elution step according to the protocol for the kit you are using. DNase I Digestion Protocol for Purified RNA in Solution This alternative protocol is for the removal of genomic DNA from RNA that has already been eluted and not previously DNase I treated. Set up the following reaction: RNA sample: 90 μl Solution D1: 10 μl DNase I stock enzyme: 2 μl Sterile water: bring the volume up to 100 μl Mix and incubate at room temperature ( C) for 10 minutes. Inactivate DNase I by adding EDTA to a final concentration of 5 mm to chelate the divalent cations then heat to 75 0 C for 5 minutes. Thank you for choosing the On-Spin Column DNase I (RNase-Free)! 4

5 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 5

6 Other Quality Products Available from MO BIO Laboratories, Inc. For more product and detailed information go to to request a catalog. DNA Purification and Gel Extraction Catalog No. Quantity Genomic DNA Isolation Continued Catalog No. Quantity PowerClean DNA Clean-Up PowerSoil-htp 96 Well Soil DNA Isolation UltraClean 15 DNA Purification preps UltraClean Soil DNA Isolation UltraClean PCR Clean-Up UltraClean-htp 96 Well Soil DNA Isolation UltraClean-htp 96 Well PCR Clean- Up UltraClean Mega Soil DNA Isolation preps UltraClean GelSpin DNA Extraction Plasmid DNA Isolation Catalog No. Quantity UltraClean 6 Minute Mini Plasmid Prep UltraClean Standard Mini Plasmid Prep PowerClean DNA Clean-Up UltraClean Fecal DNA Isolation PowerMicrobial Midi DNA Isolation preps PowerMicrobial Maxi DNA Isolation preps UltraClean-htp 96 Well Plasmid Prep UltraClean Microbial DNA Isolation UltraClean Midi Plasmid Prep preps UltraClean-htp 96 Well Microbial DNA Isolation UltraClean Maxi Plasmid Prep preps 20 preps PowerPlant DNA Isolation UltraClean Endotoxin-Free Mini Plasmid Prep UltraClean Plant DNA Isolation UltraClean Endotoxin-Free Midi Plasmid Prep preps UltraClean-htp 96 Well Plant DNA Isolation UltraClean Endotoxin-Free Maxi Plasmid Prep preps UltraClean Tissue & Cells DNA Isolation UltraClean Endotoxin Removal kit UltraClean-htp 96 Well Tissue DNA Isolation UltraClean Endotoxin-Free Ethanol kit UltraClean Blood DNA Isolation Precipitation (Non-Spin) UltraClean Endotoxin Removal ml UltraClean Blood DNA Isolation kit Reagent (Processes 1,000 ml of Blood) Endotoxin-Free Sodium Chloride ml UltraClean Blood DNA Isolation kit Plus RNase (Processes 1,000 ml of Blood) Endotoxin-Free Centrifuge Tubes each/2 ml UltraClean BloodSpin DNA Isolation each/15 ml 25 each/50 ml RNA Isolation Catalog No. Quantity UltraClean-htp 96 Well BloodSpin DNA Isolation RNA PowerSoil Total RNA Isolation preps UltraClean Mega BloodSpin DNA preps Isolation UltraClean Microbial RNA Isolation UltraClean Forensic DNA Isolation isolations 20 isolations UltraClean Tissue RNA Isolation UltraClean Water DNA Isolation (0.45µm filters) preps 25 preps UltraClean Plant RNA Isolation preps UltraClean Water DNA Isolation (0.22 µm filters) preps 25 preps Genomic DNA Isolation Catalog No. Quantity UltraClean Water DNA Isolation (no filters) NF NF 10 preps 25 preps PowerMax Soil DNA Isolation preps PowerSoil DNA Isolation

7 Other Quality Products Available from MO BIO Laboratories, Inc. For more product and detailed information go to to request a catalog. Microbiological Culture Media Catalog No. Quantity TB DRY Powder Growth Media LB Broth Powder Growth Media, ph 7 LB Agar Powder Growth Media, ph 7 LB Broth (Lennox) Powder Growth Media, ph 7 LB Agar (Lennox) Powder Growth Media, ph 7 Soybean-Casein Digest Medium (TSB), USP Soybean-Casein Digest Agar Medium (TSA), USP Yeast Extract Tryptone Agar, Bacteriological Grade Other Reagents and Lab Accessories Continued Catalog No. Quantity Ethidium Bromide Destaining Tea bags Bags Bromophenol Blue Gel Loading Buffer Bromophenol Blue/Xylene Cyanol Gel Loading Buffer TAE Buffer, 50X (Tris-acetate-EDTA) TBE Buffer, 10X (Tris-borate-EDTA) RNase-Free Gloves 1555-XS 1555-S 1555-M 1555-L UltraClean Lab Cleaner OmniTaq DNA Polymerase Enzyme OmniTaq DNA Polymerase 2x Master Mix Omni KlenTaq DNA Polymerase Enzyme ml 5 x 1 ml 1 ml 5 x 1 ml 100 ml 500 ml 1 liter 100 ml 500 ml 1 liter bag of 100 bag of 100 bag of 100 bag of ml squeeze bottle 500 ml spray bottle 1 liter bottle reactions (10 U/µl) reactions (5 x 1.25 ml/tube) reactions (25 U/µl) Omni KlenTaq DNA Polymerase 2x Other Reagents and Lab Master Mix Accessories Catalog No. Quantity 20 bp DNA Ladder µg DNase (RNase-Free) mg 100 bp DNA Ladder µg Proteinase K kb DNA Ladder µg Ribonuclease A UltraClean Agarose, Molecular Biology Grade UltraClean MS-8 Agarose UltraClean Forensic Agarose UltraClean Low Melt Agarose UltraClean Low Melt Sieve Agarose Ethidium Bromide Solution ml 10 ml reactions (5 x 1.25 ml/tube) PCR Water Molecular Biology Grade Water DEPC Treated Water Endotoxin-Free Water mg 2 ml (20 mg/ml) 1 ml (25 mg/ml) 5 ml (25 mg/ml) 1 ml 5 x 1 ml 10 x 1 ml 10 ml bottle 200 ml 5 x 200 ml 200 ml 5 x 200 ml 10 ml 50 ml 100 ml 500 ml

8 Other Quality Products Available from MO BIO Laboratories, Inc. For more product and detailed information go to to request a catalog. Instrumentation and Accessories Catalog No. Quantity Instrumentation and Accessories Continued Catalog No. Quantity Precellys 24 Homogenizer, 120V unit Vortex Adapter, holds 2 (50 ml) V unit Ceramic Bead Tubes, 1.4 mm bead Vortex Adapter, holds 24 ( ml) V unit Ceramic Bead Tubes, 2.8 mm bead Power Supply w/timer, (120V) unit Glass Bead Tubes, 0.5 mm bead Power Supply w/timer, (220V) unit Glass Bead Tubes, 0.1 mm bead Polycarbonate Single-sided Comb Metal Bead Tubes, 2.38 mm bead Polycarbonate Dual-sided Comb ml Tough Tubes with Cap x 2.0 ml Tough Tubes with Cap 1000 x 2.0 ml Tough Tubes with Cap Carbide Bead Tubes, 0.25 mm x 0.5 ml Garnet Bead Tubes, 0.15 mm x 0.5 ml Garnet Bead Tubes, 0.70 mm x 2 ml 2 ml Collection Tubes T T T ml Screw Cap Tubes E 200 & caps Teflon Single-sided Comb mm x 3 well 1 mm x 8 well 1 mm x 10 well 1 mm x 12 well 1 mm x 8 well/16 well 1 mm x 10 well/14 well 2 mm x 8 well/16 well 2 mm x 10 well/14 well 1 mm x 3 well 1 mm x 8 well 1 mm x 10 well 1 mm x 12 well Teflon Dual-sided Comb mm x 8 well/16 well 1 mm x 10 well/14 well 2 mm x 8 well/16 well 2 mm x 10 well/14 well Mini Horizontal Gel System each Mini Horizontal Gel Caster, 1 place each Mini Horizontal Gel Caster, 3 place each Mini Horizontal Gel Tray each 15 ml Collection Tubes T Well Plate Shaker (120V) unit 50 ml Centrifuge Tubes T Well Plate Shaker (220V) unit Spin Filters SF 50 filters in Plate Adapter Set set SF SF 100 filters in 250 filters in Endotoxin-Free Centrifuge Tubes each/2 ml Tube Adapter Set set each/15 ml 25 each/50 ml 15 ml Midi Spin Filters SF 25 spin filters Vacuum Pump (120V) unit Vortex-Genie 2 Vortex (120V) V 1 unit Vacuum Pump (220V) unit Vortex-Genie 2 Vortex (220V) V unit UltraVac Manifold unit Vortex Adapter, holds 12 ( ml) V1 1 unit Vortex Adapter, holds 4 (15 ml) V unit 8