PureSpin Gel DNA Purification Kit Micro Columns INSTRUCTION MANUAL KIT COMPONENTS For Research Use Only PureSpin Gel DNA Purification Kit, Micro Columns w/out Caps (Kit Size) OD2030 (50 Preps) OD2030-2 (200 Preps) Storage Temperature Agarose Dissolving Buffer 50ml 2x100ml Room Temperature DNA Wash Buffer 1 (concentrate) 6ml 24ml Room Temperature DNA Elution Buffer 1ml 4ml Room Temperature DNA PureSpin Micro Columns 50 200 Room Temperature Collection Tubes 50 200 Room Temperature
FEATURES Quick, 15 minute high-yield recovery of ultra-pure DNA from agarose gels. Innovative PureSpin Micro Column design. Minimal elution volumes ( 6μl) and complete sample recovery with no carryover. Eluted DNA is well suited for use in DNA ligation, sequencing, labeling, PCR, etc. CONTENTS Contents p. 2 Specifications p. 3 Product Description p. 4 Buffer Preparation p. 5 Protocols p. 5 Troubleshooting p. 6 Ordering Information p. 8 Related Products p. 8 Notes from the cover: Integrity of kit components is guaranteed for up to one year from date of purchase. Reagents are routinely tested on a lot-to-lot basis to ensure they provide the highest performance and reliability. 1 Ethanol must be added prior to use as indicated on the DNA Wash Buffer label. Page 2
SPECIFICATIONS Sample Sources: DNA Purity: DNA Size Limits: DNA Recovery: Product Detergent Tolerance: Equipment Needed: Format: Purify DNA from agarose gel. Pure DNA is especially well suited for sequencing and ligation reactions. Approximately 50bp to 23kb. Usually up to 5µg total DNA per column can be eluted into as little as 6µl of low salt DNA Elution Buffer or water. For DNA 50bp to 10kb, the recovery is 70-90%. For DNA 11kb to 23kb, the recovery is 50-70%. 5% Triton X-100, 5% Tween-20, 5% Sarkosyl, 0.1% SDS. Microcentrifuge, vortex. DNA PureSpin Micro Column Notes: This product is for research use only and should only be used by trained professionals. It is not intended for use in diagnostic procedures. Some reagents included with this kit are irritants. Wear protective gloves and eye protection. Follow the safety guidelines and rules enacted by your research institution or facility. Page 3
Page 4 PRODUCT DESCRIPTION The OmniGenX PureSpin Gel DNA Purification Kit quickly and easily purifies DNA from agarose gels. Start by adding the Agarose Dissolving Buffer to the gel slice containing your DNA sample and then transfer to the supplied DNA PureSpin Micro Column. There is no need to add any organic denaturants or chloroform. The PureSpin column technology purifies DNA in just 15 minutes and is perfectly suited for use in DNA ligation reactions, sequencing, DNA labeling reactions, PCR, etc. Pvull Recovered by digestomnigenx Kit Blunt-End Ligation 0 30 60 M 1 2 3 4 5 3.0 kb - 330 bp - Blunt-end ligation of DNA fragments purified using the OmniGenX PureSpin Gel DNA Purification Kit. Fragments from plasmid DNA digested with Pvu II restriction endonuclease were purified, then mixed and ligated for the indicated times. Effectiveness of the OmniGenX PureSpin Gel DNA Purification Kit. Lanes: M: DNA Ladder; 1-5: DNA from ladder that was excised and recovered from gel. OmniGenX gel purification kits are offered in single column format (for DNA fragments up to 23kb). The OmniGenX PureSpin Gel DNA Purification Kits are also available for large DNA (up to 200kb) gel recovery. DNA PureSpin Micro Column Page 4
BUFFER PREPARATION Add 24ml 100% ethanol (26ml 95% ethanol) to the 6ml DNA Wash Buffer concentrate (OD2080-WB6). Add 96ml 100% ethanol (104ml 95% ethanol) to the 24ml DNA Wash Buffer concentrate (OD2080-WB). PROTOCOLS 1. Cut and remove the DNA fragment 1 from the agarose gel using a razor blade, scalpel or other device and transfer it into a 1.5ml microcentrifuge tube. 2. Add Agarose Dissolving Buffer in a ratio of 3 : 1 to each volume of agarose excised from the gel (e.g. for 100µl (mg) of agarose gel slice add 300µl of Agarose Dissolving Buffer). 3. Proceed by incubating at 37-55 C for 5-10 minutes until the gel slice is completely dis solved 2. For DNA fragments > 8kb, following the incubation step, add one additional volume of water to the mixture for better DNA recovery (e.g., 100µl agarose, 300µl Agarose Dissolving Buffer, and 100µl water). 4. Transfer the completely dissolved agarose solution to a DNA PureSpin Micro Column in a Collection Tube. 5. Centrifuge for 30-60 seconds at 10,000-16,000 x g. Discard the flow-through 3. 6. Add 200µl of DNA Wash Buffer to the column and centrifuge for 30 seconds at 10,000-16,000 x g. Discard the flow-through. Repeat the wash step. 7. Dispense 6µl DNA Elution Buffer or water 4 directly to the column matrix. Place column into a 1.5ml tube and centrifuge for 30-60 seconds at 10,000-16,000 x g to elute DNA. Ultra-pure DNA is now ready for use. Notes: 1 The amount of agarose excised from the gel should be as small as possible. 2 Do not incubate above 60 C. It is important that the gel slice dissolve completely. This can be facilitated by gentle mixing during the incubation. 3 Remove the flow-through by aspiration. Avoid contamination of the collection tube rim. 4 Elution of DNA from the column is dependent on ph and temperature. If water is used, make sure the ph is >6.0. Waiting 1 minute prior to elution may improve the yield of larger (> 6kb) DNA. For even larger DNA (> 10kb), the total yield may be improved by eluting the DNA with 60-70 C DNA Elution Buffer. Page 5
TROUBLESHOOTING Low Recovery: Agarose is not Fully Dissolved Small globules of undissolved agarose could interfere with DNA recovery by clogging the column and leeching salts into the eluate. Gel Incubation Temperature Above 60 C DNA may be become denatured during incubation if dissolved at a higher temperature. For optimal results, dissolve the gel slice between 37-55 C. Ethanol Addition Make sure ethanol has been added to the DNA Wash Buffer concentrate. Ethanol Evaporation Cap the bottle tightly to prevent evaporation over time. Inproper Addition of Elution Buffer Add DNA Elution Buffer directly to the column matrix and not to the walls of the column. Elution buffer requires contact with the matrix for at least 1 minute for large DNA 10kb. Elution Conditions DNA elution is dependent on ph, temperature, and time. For large genomic DNA ( 50kb), apply heated elution buffer (60-70 C) and incubate for several minutes prior to elution. Multiple Elutions Additional elutions may be performed for quantitatively higher recovery but lower final DNA concentration. This is recommended for DNA 10kb. Page 6
Low A 260 /A 230 Ratios Page 7 Contamination Be sure that the tip of the column does not come into contact with the flowthrough when removing from the collection tube. Salts from the flowthrough can contaminate a sample resulting in low A 260 / A 230 ratios. Ethanol contamination from the flowthrough can also interfere with DNA elution. OmniGenX DNA PureSpin Micro columns are designed for complete elution with no buffer retention or carryover. Supplier Q Column design causes 2-3μl CARRYOVER OmniGenX DNA PureSpin Column design ensures NO CARRYOVER Multiple Bands Appear in an Agarose Gel Use of DNA Loading Dye If water is used to elute the DNA, 6X Loading Dye containing 1mM EDTA is recommended. Most loading dyes do not contain EDTA and will acidify (ph 4) over time due to some microbial growth. This low ph is enough to cause DNA degradation. Page 7
www.omnigenx.com info@omnigenx.com ORDERING INFORMATION Product Description Catalog No. Kit Size (Preps.) PureSpin Gel DNA Purification Kit, Micro Columns w/out Caps OD2030 OD2030-2 50 200 RELATED PRODUCTS For Individual Sale Catalog No. Packaging Agarose Dissolving Buffer OD2030-DB 50ml DNA Wash Buffer (concentrate) OD2080-WB 24ml DNA Elution Buffer OD2080-EB 50ml DNA PureSpin Micro Columns, Uncapped DNA PureSpin Micro Columns, Capped Collection Tubes OX1010 OX1010-250 OX1030 OX1030-250 OX1350 OX1350-1 50 columns 250 columns 50 columns 250 columns 50 tubes 1000 tubes DISTRIBUTED BY E&K Scientific Products, Inc. Phone 800.934.8114 Fax 408.567.9671 E-mail service@eksci.com Website www.eksci.com