UltraClean Blood DNA Isolation Kit (Non-Spin)

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1 UltraClean Blood DNA Isolation (Non-Spin) Catalog No. Quantity Preps (300 μl each) Instruction Manual Please recycle Version:

2 Table of Contents Introduction... 3 Protocol Overview... 3 Equipment Required... 4 Contents & Storage... 4 Precautions & Warnings... 4 Protocol... 5 Hints & Troubleshooting Guide... 6 Contact Information... 7 Other Quality Products Available

3 Introduction The UltraClean Blood DNA Isolation (Non-Spin) is designed to isolate very high molecular weight genomic DNA from whole blood, buffy coat or cultured cells. It is a solution-based kit that employs a gentle procedure to eliminate shearing. This UltraClean Blood DNA Isolation provides all the reagents and protocols for isolating high quality genomic, mitochondrial or viral (if associated with white blood cells) DNA from 300µl, 3 ml and up to 10 ml of mammalian whole blood, buffy coat, bone marrow cells, culture cells, buccal swabs. Use this kit for isolating genomic DNA from 300µl of whole blood. This kit yields DNA ready for any application. Protocol Overview Lysis buffer is added directly to whole blood (fresh, frozen or stored at 4ºC). Next, cell nuclei and mitochondria are pelleted by centrifugation. The pellet is resuspended and incubated in a detergent solution, salt solution and RNase A to efficiently remove contaminants such as proteins and RNA. DNA is then precipitated by addition of isopropanol, recovered by centrifugation, washed in 70% ethanol, dried and resupended in hydration buffer. This kit is for research purposes only. Not for diagnostic use. Other Related Products Catalog No. Quantity BiOstic Bacteremia DNA Isolation UltraClean PCR Clean-Up UltraClean BloodSpin DNA Isolation

4 Equipment Required Microcentrifuge (13,000 x g) 65 C water bath Pipet (volumes required 1.5 l l) Vortex-Genie 2 Vortex (MO BIO Catalog# V or V-220) Contents: Sufficient reagents and for of 300 l blood samples Catalog # Component Catalog # Amount Solution G ml Solution G ml Solution G ml Solution G ml 100% Isopropanol ml 70% Ethanol ml RNase A ml 2 ml Collection Tubes T 200 Storage Store RNase at 4 C. All other 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. 4

5 Protocol Please wear gloves at all times 1. Add 300 µl of whole blood to 900 µl of Solution G1. 2. Invert twice and incubate 5 minutes at room temperature. 3. Invert two times during incubation. 4. Centrifuge for 30 seconds at 13,000 x g. Remove supernatant with narrow pipet tip and discard without disturbing white pellet. (About 20 µl of supernatant will remain.) 5. Bump vortex to resuspend pellet completely. 6. Check Solution G2. If precipitated, heat to C for 5 minutes to dissolve. Add 300 µl of Solution G2. 7. Pipet up and down to lyse the cells (If cell clumps are still visible incubate at 37 C until no clumps are visible). 8. Add 1.5 µl of RNase A, invert 5 times and vortex on low speed for 5 seconds. 9. Add 100 µl of Solution G Immediately vortex on high for 15 seconds. 11. Centrifuge 3 minutes at 13,000 x g. 12. Transfer the supernatant to a clean 2.0 ml Collection Tube. 13. Add 300 µl of 100% Isopropanol. 14. Invert 15 times and incubate at room temperature for 3 minutes. 15. Centrifuge 1 minute at 13,000 x g. 16. Carefully pour off supernatant without disturbing pellet, and drain on a paper towel or absorbent material. 17. Add 300 µl of 70% Ethanol. 18. Invert tube 5 times to wash the pellet. 19. Centrifuge 30 seconds at 13,000 x g. 20. Carefully pour off supernatant without disturbing pellet, and drain on a paper towel or absorbent material. 21. Centrifuge for 30 seconds, and remove residual supernatant with narrow pipet tip without disturbing pellet. 22. Add 100 µl of Solution G Heat in a 65 C water bath or heat block tapping the tube occasionally, until pellet has resuspended (should take no longer than minutes). 24. Genomic DNA in tube is now ready to use for any application. Thank you for choosing the UltraClean Blood DNA Isolation (Non-Spin). 5

6 Hints & Troubleshooting Guide Concentrating the DNA Normally the final volume will be 100 l. If this is too dilute for your purposes, you may resuspend the pellet in desired volume of Solution G4. The smaller the volume the longer it may take to re-hydrate. Amount to process 300 µl of Whole blood (or Bone Marrow). No protein pellet Sample was not cooled to room temperature before adding Solution G3. Ice the sample for 3 more minutes and centrifuge for 3 minutes. Pellet should be very visible. Another cause is Solution G3 was not thoroughly mixed when adding to sample. Always mix completely by vortexing for 15 seconds. DNA pellet was difficult to resuspend This is caused if the DNA was over-dried or was not mixed occasionally during 65 C incubation time. To solve this, heat for 1 hour at 65 C and leave sample overnight at room temperature. Low recovery yield There could be many causes to this. One is the sample contained few white blood cells. Another cause could be the white blood cell pellet was not completely resuspended. You must thoroughly resuspend pellet in step 5. If the samples were stored improperly or for too long of a period, this will effect the size and yield of DNA recovered. Always use fresh samples. You must be very careful in steps 16 and 20. The pellet is not very stable and can be lost. 6

7 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 7

8 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 RNA Isolation Continued Catalog No. Quantity PowerClean DNA Clean-Up RNA PowerSoil DNA Elution preps Accessory UltraClean 15 DNA Purification preps RNA PowerSoil Total RNA Isolation preps UltraClean PCR Clean-Up UltraClean Microbial RNA Isolation UltraClean -htp 96 Well PCR Clean- Up x 96 preps 12 x 96 preps UltraClean Tissue & Cells RNA Isolation UltraClean GelSpin DNA Extraction UltraClean Plant RNA Isolation preps Plasmid DNA Isolation Catalog No. Quantity Genomic DNA Isolation Catalog No. Quantity UltraClean 6 Minute Mini Plasmid Prep PowerLyzer PowerSoil DNA Isolation UltraClean Standard Mini Plasmid Prep UltraClean -htp 96 Well Plasmid Prep UltraClean Midi Plasmid Prep x 96 preps 12 x 96 preps 20 preps PowerLyzer UltraClean Microbial DNA Isolation PowerBiofilm DNA Isolation PowerFood Microbial DNA Isolation UltraClean Maxi Plasmid Prep preps BiOstic Bacteremia DNA Isolation preps UltraClean Endotoxin-Free Mini BiOstic FFPE Tissue DNA Isolation Plasmid Prep UltraClean Endotoxin-Free Midi preps BiOstic Paraffin Removal Reagent x 25 ml Plasmid Prep UltraClean Endotoxin-Free Maxi preps PowerMax Soil DNA Isolation preps Plasmid Prep UltraClean Endotoxin Removal kit PowerSoil DNA Isolation UltraClean Endotoxin-Free Ethanol Precipitation kit PowerSoil -htp 96 Well Soil DNA Isolation x 96 preps 12 x 96 preps UltraClean Endotoxin Removal Reagent ml UltraClean Soil DNA Isolation Endotoxin-Free Sodium Chloride ml UltraClean -htp 96 Well Soil DNA Isolation x 96 preps 12 x 96 preps Endotoxin-Free Centrifuge Tubes each/2 ml UltraClean Mega Soil DNA Isolation preps each/15 ml 25 each/50 ml RNA Isolation Catalog No. Quantity PowerClean DNA Clean-Up PowerLyzer UltraClean Tissue & Cells RNA Isolation UltraClean Fecal DNA Isolation PowerLyzer UltraClean Plant RNA PowerMicrobial Midi DNA Isolation preps Isolation PowerBiofilm RNA Isolation PowerMicrobial Maxi DNA Isolation preps LifeGuard Soil Stabilization Solution On-Spin Column DNase I (RNase- Free) BiOstic Stabilized Blood RNA Isolation BiOstic Blood Total RNA Isolation 10 ml 100 ml 1 L 7.5 L UltraClean Microbial DNA Isolation UltraClean -htp 96 Well Microbial DNA Isolation preps preps PowerPlant DNA Isolation UltraClean Plant DNA Isolation x 96 preps 12 x 96 preps 2 8

9 Other Quality Products Available from MO BIO Laboratories, Inc. For more product and detailed information go to to request a catalog. Genomic DNA Isolation Continued Catalog No. Quantity UltraClean -htp 96 Well Plant DNA x 96 preps Isolation x 96 preps UltraClean Tissue & Cells DNA Isolation UltraClean -htp 96 Well Tissue DNA Isolation UltraClean Blood DNA Isolation (Non-Spin) UltraClean Blood DNA Isolation (Processes 1,000 ml of Blood) UltraClean Blood DNA Isolation Plus RNase (Processes 1,000 ml of Blood) UltraClean BloodSpin DNA Isolation UltraClean -htp 96 Well BloodSpin DNA Isolation UltraClean Forensic DNA Isolation PowerWater DNA Isolation RapidWater DNA Isolation UltraClean Water DNA Isolation (0.45µm filters) UltraClean Water DNA Isolation (0.22 µm filters) UltraClean Water DNA Isolation (No filters) x 96 preps 12 x 96 preps Other Reagents and Lab Accessories Catalog No. Quantity LB Agar (Lennox) Powder Growth Media, ph Soybean-Casein Digest Medium (TSB), USP Soybean-Casein Digest Agar Medium (TSA), USP Yeast Extract kit Tryptone kit Agar, Bacteriological Grade x 96 preps x 96 preps isolations isolations NF NF NF NF NF NF (No filters) (0.22 μm) (0.45 μm) (No filters) (0.22 μm) (0.45 μm) (No filters) (0.22 μm) (0.45 μm) (No filters) (0.22 μm) (0.45 μm) 10 preps 25 preps 10 preps 25 preps 10 preps 25 preps Microbiological Culture Media Catalog No. Quantity TB DRY Powder Growth Media LB Broth Powder Growth Media, ph LB Agar Powder Growth Media, ph LB Broth (Lennox) Powder Growth Media, ph bp DNA Ladder µg 100 bp DNA Ladder µg 1 kb DNA Ladder µg UltraClean Agarose, Molecular Biology Grade UltraClean MS-8 Agarose UltraClean Forensic Agarose UltraClean Low Melt Agarose UltraClean Low Melt Sieve Agarose Ethidium Bromide Solution Ethidium Bromide Destaining Tea Bags Bromophenol Blue Gel Loading Buffer Bromophenol Blue/Xylene Cyanol Gel Loading Buffer 50 g 100 g 50 g 100 g 50 g 100 g 50 g 100 g 50 g 100 g 1 ml 10 ml bags TAE Buffer, 50X (Tris-acetate-EDTA) ml 5 x 1 ml 1 ml 5 x 1 ml 100 ml 500 ml 1 liter 9

10 Other Quality Products Available from MO BIO Laboratories, Inc. For more product and detailed information go to to request a catalog. Other Reagents and Lab Accessories Continued Catalog No. Quantity Instrumentation and Accessories Catalog No. Quantity TBE Buffer, 10X (Tris-borate-EDTA) ml 500 ml 1 liter PowerLyzer 24 Bench Top Bead- Based Homogenizer (110/220V) unit RNase-Free Gloves 1555-XS 1555-S 1555-M 1555-L UltraClean Lab Cleaner KAPA PROBE FAST qpcr s KAPA SYBR FAST Universal 2X qpcr Master Mix KAPA2G Robust HotStart ReadyMix bag of 100 bag of 100 bag of 100 bag of ml squeeze bottle 500 ml spray bottle 1 liter bottle 100 reactions 500 reactions 1000 reactions 100 reactions 500 reactions 1000 reactions 100 reactions 500 reactions PowerLyzer Tube Holder unit PowerLyzer Tube Holder Stand unit PowerVac Mini System unit + 20 adapters PowerVac Manifold unit PowerVac Mini Spin Filter Adapters adapters 20 adapters KAPA HiFi HotStart ReadyMix reactions Ceramic Bead Tubes, 1.4 mm bead reactions KAPA2G FAST HotStart DNA reactions Ceramic Bead Tubes, 2.8 mm bead Polymerase with dntps reactions 500 reactions KAPA2G FAST HotStart ReadyMix reactions Glass Bead Tubes, 0.5 mm bead reactions KAPA Long Range HotStart DNA reactions Glass Bead Tubes, 0.1 mm bead Polymerase with dntps reactions 500 reactions KAPA Taq Polymerase ReadyMix reactions Metal Bead Tubes, 2.38 mm bead OmniTaq DNA Polymerase Enzyme reactions (10 U/µl) OmniTaq DNA Polymerase 2x reactions Master Mix (5 x 1.25 ml/tube) Omni KlenTaq DNA Polymerase reactions Enzyme (25 U/µl) Omni KlenTaq DNA Polymerase 2x reactions Master Mix (5 x 1.25 ml/tube) DNase (RNase-Free) mg units Proteinase K mg ml (20 mg/ml) Ribonuclease A (25 mg/ml) PCR Water Molecular Biology Grade Water DEPC Treated Water Endotoxin-Free Water ml 5 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 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 Garnet + ¼ Ceramic 15 ml Bead Tubes, 0.70 mm Garnet + ¼ Ceramic 50 ml Bead Tubes, 0.70 mm ` Glass 15 ml Bead Tubes, 0.1 mm Glass 50 ml Bead Tubes, 0.1 mm Glass 15 ml Bead Tubes, 1.0 mm Ceramic 15 ml Bead Tubes, 1.4 mm Ceramic 50 ml Bead Tubes, 1.4 mm Metal 50 ml Bead Tubes, 2.38 mm

11 Other Quality Products Available from MO BIO Laboratories, Inc. For more product and detailed information go to to request a catalog. Instrumentation and Accessories Continued Catalog No. Quantity Instrumentation and Accessories Continued Catalog No. Quantity PowerMix 15 ml Bead Tubes Whirl-Pak Collection Bag, Medium (1,627 ml) bags PowerMix 50 ml Bead Tubes Whirl-Pak Collection Bag, Large (3,637 ml) bags 2 ml Collection Tubes T T T ml Screw Cap Tubes E 200 & caps Whirl-Pak Stand up Bag, Small (118 ml) Whirl-Pak Stand up Bag, Medium (532 ml) bags bags 15 ml Collection Tubes T 25 Whirl-Pak Stand up Bag, Large (1,242 ml) 50 ml Centrifuge Tubes T 25 Whirl-Pak Stand up Bag, Extra- Large (2,041 ml) bags bags Spin Filters (in 1.9 ml ) SF SF SF Endotoxin-Free Centrifuge Tubes filters 100 filters 250 filters 100 each/2 ml 50 each/15 ml 25 each/50 ml Whirl-Pak Scoop Bag, 60 ml bags Anti-Static Funnels, Micro Pack of ml Midi Spin Filters SF 25 spin filters Anti-Static Funnels, Small Pack of 50 Vortex-Genie 2 Vortex (120V) V 1 unit Anti-Static Funnels, Medium Pack of 50 Vortex-Genie 2 Vortex (220V) V unit Anti-Static Funnels, Large Pack of 20 Vortex Adapter, holds 12 ( ml) V1 1 unit Mini Horizontal Gel System each Vortex Adapter, holds 6 (5 ml) V1-5 1 unit Mini Horizontal Gel Caster, 3 place each Vortex Adapter, holds 4 (15 ml) V unit Mini Horizontal Gel Tray each Vortex Adapter, holds 2 (50 ml) V unit Polycarbonate Single-sided Comb Vortex Adapter, holds 24 ( ml) V unit Polycarbonate Dual-sided Comb BagMixer 400 VW unit Teflon Single-sided Comb BagFilter 400 P Box of 500 Teflon Dual-sided Comb BagPage Box of 500 Power Supply w/timer, (120V) unit 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 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 11

12 Other Quality Products Available from MO BIO Laboratories, Inc. For more product and detailed information go to to request a catalog. Instrumentation and Instrumentation and Accessories Continued Catalog No. Quantity Accessories Continued Catalog No. Quantity 96 Well Plate Shaker (120V) unit Vacuum Pump (120V) unit 96 Well Plate Shaker (220V) unit Vacuum Pump (220V) unit Plate Adapter Set set 12

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