USB PrepEase Gel Extraction Kit

Transcription

1 USB PrepEase Gel Extraction Kit Product number 78756, 50 preps Product number 78757, 250 preps Affymetrix, USB and PrepEase are registered trademarks of Affymetrix, Inc. Genetic Performance Certified is a trademark of Affymetrix, Inc. PrepEase products are covered under European Patent EP and US Patent 6,428,703. Storage Store at room temperature (20 25 C). Warning: For research use only. Not recommended or intended for diagnosis of disease in humans or animals. Do not use internally or externally in humans or animals Affymetrix, Inc. All rights reserved. Affymetrix, Inc Miles Road Cleveland, Ohio USA P 78755A usb.affymetrix.com rev 10/11

2 Contents Components supplied...3 Storage and preparation...3 Materials not supplied...3 Quality control...4 Safety warnings and precautions...4 Introduction...5 Overview...5 Kit specifications...5 Protocols...6 DNA extraction from agarose gels...6 Direct purification of PCR samples...7 Concentration, desalination and/or removal of enzymes...7 Troubleshooting...8 References...9 Related products...10 Contact information...11 Components supplied Always keep buffer bottles tightly closed, especially if buffers are warmed prior to use. PrepEase Gel Extraction Kit 50 preps 250 preps NT Buffer 2 x 25 ml 2 x 120 ml NT3 Buffer (concentrate) 2 x 7 ml 40 ml NE Buffer 15 ml 50 ml PrepEase Cleanup Columns PrepEase Collection Tubes Storage and preparation Kit components can be stored at room temperature (20-25 C). NT3 Buffer: Add the indicated volume (see below) of 100% ethanol to the NT3 Buffer. Store NT3 Buffer at room temperature (20-25 C). Keep the bottle tightly closed. 50 preps 250 preps NT3 Buffer 2 x 7 ml 40 ml Ethanol (not supplied) 2 x 28 ml 160 ml Materials not supplied 100% Ethanol Agarose - MB Grade [PN 75817] or Agarose, Low Melt [PN 32829/30] TAE Buffer, [PN 75904] TE Buffer, ph 8.5 Nuclease-Free Water [PN 71786] 2 3

3 Quality control The PrepEase Gel Extraction Kit contains PrepEase Cleanup Columns and the necessary reagents to purify DNA from agarose gels, PCR reactions, or other enzymatic reactions. All PrepEase Cleanup Columns and reagents are free of contaminating DNase. and RNase. Safety warnings and precautions Warning: For research use only. Not recommended or intended for diagnosis of disease in humans or animals. Do not use internally or externally in humans or animals. Caution: All chemicals should be considered as potentially hazardous. We, therefore, recommend that this product is handled only by those persons who have been trained in laboratory techniques and that it is used in accordance with the principles of good laboratory practice. Wear suitable protective clothing, such as lab coat, safety glasses, and gloves. Care should be taken to avoid contact with skin and eyes. In the case of contact with skin or eyes, wash immediately with water. See MSDS (Material Safety Data Sheet) for specific advice. Introduction Overview The PrepEase Gel Extraction Kit may be used to purify DNA from agarose gels or PCR reaction mixtures. In addition, the kit may be used for desalination, and removal of enzymes, nucleotides or labeling reagents from sample DNA. Typical recovery rates range from 70-95% for DNA fragments between 50-10,000 bp. The purified DNA may be used directly in applications such as DNA sequencing, PCR, cloning, or probe labeling. With the PrepEase cleanup method, DNA binds in the presence of chaotropic salts to the silica membrane within the spin column. Contaminants such as salts and soluble macromolecular components are removed with a simple washing step. Pure DNA is eluted under low ionic strength conditions with slightly alkaline buffer. Kit specifications Parameters PrepEase Gel Extraction Kit DNA fragments from gels up to 400 mg of gel slice (< 2% agarose) PCR product purification up to 1-15 µg PCR product Desalination, removal of enzymes,. nucleotides and/or labeling. components such as biotin up to 15 µg DNA per reaction Binding capacity 15 µg Elution volume µl Prep time 10 minutes 4 5

4 Protocols Protocol for DNA extraction from agarose gels 1. Excise DNA fragment a. Run the DNA fragment(s) to be purified in either a standard or low melt agarose gel using TAE Buffer. Stain the gel with a fluorescent dye. b. Visualize the DNA fragment under U.V. illumination. Minimize exposure to U.V. light to avoid degradation of DNA. c. Excise the DNA fragment from the agarose gel using a clean, nuclease-free scalpel or razor blade. Cut off excess agarose to minimize the gel volume. d. Weigh the gel slice using a nuclease-free technique. Transfer the gel slice to a clean tube. 2. Gel solubilization a. For each 100 mg agarose gel slice add 200 μl NT Buffer. b. Incubate sample at 50 C for 5-10 minutes or until the gel is completely dissolved. c. Vortex the sample briefly every 2-3 minutes to aid the process. The maximum weight of gel slice per PrepEase Cleanup Column is 400 mg for gels containing less than 2% agarose. 3. Bind DNA sample to column a. Place a PrepEase Cleanup Column into a 2 ml PrepEase Collecting Tube. b. Load the sample directly to the center of the cleanup column. c. Centrifuge for 1 minute at 11,000 x g. d. Discard flow-through and place the cleanup column back into the. collecting tube. 4. Wash column a. Add 600 µl NT3 Buffer directly to the cleanup column. b. Centrifuge for 1 minute at 11,000 x g. c. Discard flow-through and place the cleanup column back into the. collecting tube. 5. Dry column Centrifuge for 2 minutes at 11,000 x g to remove excess NT3 Buffer. Make sure the spin column doesn t come in contact with the flow-through while removing it from the centrifuge and the collecting tube. Residual ethanol from NT3 Buffer must be removed so it will not inhibit subsequent reactions. Total ethanol removal can be achieved by incubating the PrepEase Cleanup Columns for 2-5 minutes at 70 C prior to elution. 6. Elute DNA a. Place the cleanup column into a clean 1.5 ml microcentrifuge tube. b. Add µl NE Buffer. c. Incubate at room temperature for 1 minute to increase the yield of eluted DNA. d. Centrifuge for 1 minute at 11,000 x g. Yield of larger fragments (> 5-10 kb) can be increased by using prewarmed elution buffer (70 C). Incubate at room temperature for 1 minute before collecting eluate by centrifugation. Protocol for direct purification of PCR samples 1. Prepare sample for binding Mix 2 volumes of NT Buffer per 1 volume of sample (for example: 200 µl NT Buffer and 100 µl PCR product mixture). For sample volumes < 50 µl adjust the volume of the reaction mix to 50 µl using TE Buffer (ph 7.5). Then mix 2 volumes of NT Buffer with 1 volume of PCR sample. Continue with Step 3 of the protocol for DNA extraction from agarose gels. Protocol for concentration, desalination and/or removal of enzymes 1. Prepare sample for binding Add 2 volumes of NT Buffer to 1 volume of sample (for example 200 µl NT Buffer and 100 µl PCR reaction mix). If the sample contains large amounts of detergents or other substances, double the volume of NT Buffer. For sample volumes < 50 µl adjust the volume of the reaction mix to 50 µl using TE Buffer (ph 7.5). Then mix 2 volumes of NT Buffer with 1 volume of PCR sample. Continue with Step 3 of the protocol for purification of DNA from agarose gels. 6 7

5 Troubleshooting Problem Possible causes and solutions Incomplete solubilization 1. High concentrations of agarose of agarose slices.. Double the volume of NT Buffer for highly concentrated agarose gels (>2%). 2. Gel slice not completely dissolved. Ensure incubation is performed at 50 C. Depending on the weight of the gel slice, extend the incubation to 20 minutes. Vortex every. 2 minutes to aid the process. Large gel slices may be crushed before the addition of NT Buffer. Low DNA yield 1. Reagents not prepared properly. Add indicated volume of 100% ethanol to NT3 Buffer concentrate and mix well before use. 2. Gel slice not completely dissolved. Ensure incubation is performed at 50 C. Depending on the weight of the gel slice, extend the incubation to 20 minutes. Vortex every. 2 minutes to aid the process. Large gel slices may be crushed before the addition of NT Buffer. 3. Insufficient drying of the PrepEase Cleanup Column. Centrifuge 5 minutes at 11,000 x g before elution to remove NT3 Buffer completely (contains ethanol). Ethanol contamination is also indicated by gel-loading problems (samples float out of wells). Remove the cleanup column carefully from the centrifuge and collecting tube and avoid contact with flow-through. 4. Not enough elution buffer. When larger amounts of DNA (>5 µg) are bound, increase the elution buffer volume up to 100 µl. 5. Isolation of large DNA fragments. Pre-heat NE Buffer to 70 C prior to elution and allow to equilibrate on the silica membrane at room temperature for 2 minutes before centrifugation. Problem Possible causes and solutions Suboptimal performance 1. Carry-over of ethanol-containing NT3 Buffer of DNA in sequencing.. Be sure to centrifuge 3-5 minutes at 11,000 x g reactions. in order to achieve complete removal of NT3 Buffer after the washing step. Ethanol contamination is also indicated by gel-loading problems (samples float out of wells). 2. Elution of DNA with buffers other than NE Buffer. EDTA may inhibit sequencing reactions. In this case it is recommended to repurify DNA and elute in NE Buffer or water. 3. Not enough DNA used for sequencing reaction. Carefully quantify DNA sample prior to using in sequencing reactions. If problems persist please contact Technical Support for assistance at (888) or USBtechsupport@affymetrix.com. For technical support outside the U.S., please visit our website for up-to-date contact information within your area. Reference 1. Vogelstein, B. and Gillespie, D. (1979) Proc. Natl. Acad. Sci. USA, 76,

6 Related products Ultrapure Reagents Product Application Pack size Product number Agarose - Separation Gel electrophoresis 25 gm bp, Genetic. 100 gm. Performance Certified gm gm Agarose - LE Gel electrophoresis 25 gm gm gm gm Agarose - Low Melt Gel electrophoresis 25 gm Separation 1000 bp, gm. Genetic Performance. Certified Agarose - Low Melt Gel electrophoresis 25 gm Separation 1000 bp, gm. Genetic Performance. Certified Ethidium Bromide Drops Gel electrophoresis 5 ml TAE Buffer, 10X Solution Gel electrophoresis 1 L L TE Buffer, 1X Solution Storage of DNA 10 x 1 ml ml ml Water, Nuclease-Free Resuspending DNA 10 x 1 ml Buffer preparation, 100 ml. DNA storage 500 ml.. 1 L.. 5 L Markers and Ladders Product Application Pack size Product number DNA Ladder, 1 kb Plus Gel electrophoresis 500 µl DNA Ladder, 100 bp Gel electrophoresis 500 µl PCR Markers, 50-2,000 bp Gel electrophoresis 250 µl Affymetrix, Inc. usb.affymetrix.com USA. Europe Cleveland, Ohio. High Wycombe, United Kingdom (888) (216) (0) USB products distributed outside the USA:. Please visit our website at usb.affymetrix.com for up-to-date contact information within your area

7 Material Safety Data Sheet Revision: 03/10/2010 Hazard information is provided for compliance with both the. UK Chemicals (Hazard Information and Packaging) (CHIP). Regulations and the US Hazard Communication Standard (HCS) IDENTIFICATION OF THE PRODUCT NAME: PRODUCT CODE: EEC NUMBER: SUBSTANCE/PREPARATION USB PrepEase Gel Extraction Kit / / AND COMPANY (Guanidine Thiocyanate) SUPPLIER:.. EMERGENCY CONTACT: USB Products - Affymetrix, Inc. Chemtrec: (800) Miles Road, Cleveland, Ohio Phone: (216) Outside USA & Canada: Please visit our website at for contact information on USB product distributors within your area. COMPOSITION/ HAZARDOUS COMPONENTS HAZARD CAS NO. %WT TLV CHIP R & S Phrases.. Guanidine <30% R:20/21/22 Harmful by inhalation, in.. Thiocyanate... contact with skin and if swallowed. (NT Buffer) R:32 Contact with acids liberates very toxic gas S:13 Keep away from food, drink and animal feeding stuffs. HAZARDS IDENTIFICATION CHIP Harmful HCS Irritant FIRST-AID MEASURES EYES: Flush with water for 15 minutes. Seek medical advice if irritation persists. SKIN: Flush with water, then wash thoroughly with soap and water. Remove contaminated clothing and wash before reuse. Seek medical attention if irritation persists.. INHALATION: Remove the victim from exposure and move to fresh air. If breathing is difficult, give oxygen. If not breathing, give artificial respiration. Keep victim quiet and warm. Seek immediate medical attention.. INGESTION: Drink water and seek immediate medical attention. Avoid alcoholic beverages. Never give anything by mouth to an unconscious person.. ANTIDOTE: Always have a cyanide antidote kit on hand when working with cyanide compounds. Obtain medical advice on proper use. FIRE-FIGHTING INFORMATION Use media suitable to extinguish the supporting or surrounding fire. Wear NIOSH (or equivalent) approved self contained breathing apparatus. For small fires only: use carbon dioxide, dry powder or foam. Emits toxic fumes under fire conditions.. Flash Point = No data available. ACCIDENTAL RELEASE MEASURES Wear appropriate personal protective equipment and clothing including lab coat, safety glasses, gloves and NIOSH-approved respirator. Collect in a manner that does not create dust and place in a suitable waste container. Avoid contact of material with skin or eyes. Use adequate ventilation. HANDLING AND STORAGE Wear appropriate personal protective equipment and clothing including lab coat, safety glasses, gloves and NIOSH-approved respirator. Avoid contact of material with skin or eyes. Use adequate ventilation. Store ambient away from incompatible materials. PERSONAL PROTECTION Wear appropriate personal protective equipment and clothing including lab coat, safety glasses, gloves and NIOSH-approved respirator. A qualified industrial hygienist should evaluate the need for respiratory protection. Use respiratory protection approved by NIOSH (or equivalent) and appropriate to the hazard. Avoid contact of material with skin or eyes. Mechanical ventilation or local exhaust as needed to control exposure to dust, vapors or mists. Access to a safety shower and eye-wash. PHYSICAL AND CHEMICAL Appearance: Kit containing vials of solution Boiling Point: No data available PROPERTIES Vapor Pressure: No data available Vapor Density: No data available Solubility (Water): Soluble Specific Gravity: No data available. Percent Volatile: No data available Evaporation Rate: No data available. Chemical Formula: Kit STABILITY AND REACTIVITY Product is stable. Hazardous decomposition products include hydrogen cyanide, ammonia, and oxides of carbon, nitrogen and sulfur. Incompatible with iron, strong acids and strong oxidizing agents. Heating to decomposition or contact with acids or acid vapors can liberate poisonous cyanide vapors. Hazardous polymerization will not occur

8 TOXICOLOGICAL INFORMATION EFFECTS OF OVEREXPOSURE: EYES: Contact may cause irritation and slight corneal injury.. SKIN: Harmful if absorbed through skin. If absorbed, may cause symptoms similar to those for ingestion. Contact may cause irritation and/or allergic reaction.. INHALATION: Harmful if inhaled. May cause similar effects as those described under ingestion. May cause irritation to mucous membranes and upper respiratory tract. Exposure to low and even unmeasurable isocyanate concentration may result in sensitization.. INGESTION: Harmful if swallowed. Chronic ingestion or excessive dosage may cause gastrointestinal tract irritation with nausea, vomiting and diarrhea. Ingestion of thiocyanates may also cause disorientation, weakness, low blood pressure, confusion, psychotic behavior, muscular spasm, convulsions and possibly death.. TARGET ORGAN(S): Kidneys, CNS, Liver, Lungs and Cardiovascular System.. ADDITIONAL INFORMATION:. May cause liver and kidney damage. May cause lung damage. May cause central nervous system depression. Repeated or prolonged exposure may cause allergic reactions in sensitive individuals. Inhalation may aggravate existing chronic respiratory problems such as asthma, emphysema or bronchitis.. Toxicity data listed for Guanidine Thiocyanate in RTECS under XL Intraperitoneal Mouse LD50 = 300 mg/kg. Details of toxic effects not reported other than lethal dose value.. Definition(s): RTECS = Registry of Toxic Effects of Chemical Substances. ECOLOGICAL INFORMATION No information available. DISPOSAL CONSIDERATIONS Dispose of material in accordance with applicable local, state, and federal regulations. TRANSPORTATION INFORMATION US DOT / IATA: No applicable information. REGULATORY INFORMATION RCRA - No applicable information. SARA This material does not have an RQ or TPQ.. SARA This material is not reportable under EPA TSCA Section 8(b) - For Guanidine Thiocyanate: Chemical Inventory.. Exposure Limits - Not established.. California Proposition 65 - No applicable information. This data sheet is based upon information believed to be reliable. The Company makes no statement or warranty as to the accuracy or. completeness of the information contained herein which is offered for your consideration, investigation and verification. Any use of the. information contained in this data sheet must be determined by the user to be in accordance with appropriate applicable regulations. 14