Concert 96 Protein Screen - Thermo Fisher Scientific
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1. PCR Micro Kits contains Binding Buffer B2 a proprietary blend allowing for routine purifications of 125 bp 12 3 kb dsDNA PCR fragments Continued on next page Overview Continued Advantages Downstream Applications PureLink Micro Kit Column Kit Specifications The advantages of using the PureLink PCR Micro Kit are e High DNA concentrations in low elution volumes e Efficient removal of primers dNTPs salts and enzymes without the need to perform ethanol precipitation e Efficient removal of unincorporated dye labeled nucleotides from cDNA labeling reactions e Minimal variation in elution volume recovery e Purifies PCR products in 6 minutes e Excellent performance of the purified PCR products in downstream applications The purified PCR product is suitable for any downstream application including e DNA sequencing e Cloning e Restriction enzyme digestion e PCR reactions e Labeling Binding Capacity 5 ug dsDNA Column Reservoir Capacity 800 ul Collection Tube Capacity 2 0 ml Elution Tube Capacity 1 7 ml Centrifuge Compatibility Capable of centrifuging gt 14 000 x g Starting Material 50 100 ul PCR product Elution Volume 10 ul Separation Range 0 1 12 kb from 10 40 mer primers DNA Recovery up to 95 Primer Removal gt 95 Processing time 6 minutes Continued on next page 2 Overview Continued Workflow The flow chart below illustrates the steps for purifying you2. room temperature Upon receipt store all contents at room temperature Kit contents are guaranteed stable for six months when properly stored The components included in the PureLink PCR Micro Kits are described below Sufficient reagents are provided to perform 50 preps for catalog number K310050 and 250 preps for catalog number K310250 Component Quantity K310050 K310250 Binding Buffer B2 72ml Wash Buffer W1 40 ml Elution Buffer E5 15ml 10 mM Tris HCl pH 8 5 PureLink Micro Kit Columns 5 x 50 each with Collection Tubes PureLink Elution Tubes 5 x 50 each Accessory Products Additional The following products are also available from Invitrogen Products For more details visit our web site at www invitrogen com or contact Technical Support page 10 Product Catalog No PureLink PCR Micro Kit 10 prep K310010 PureLink PCR Purification Kit 50 preps K3100 01 250 preps K3100 02 PureLink 96 PCR Purification Kit K3100 96 PureLink Quick Gel Extraction Kit 50 preps K2100 12 250 preps K2100 25 UltraPure DNase RNase Free Distilled Water 500 ml 10977 015 Quant iT DNA Assay Kit High Sensitivity E Gel E Gel Agarose Gels are bufferless pre cast agarose gels Agarose Gels designed for fast convenient electrophoresis of DNA and DNA samples E Gel agarose gels are available in different Ladders agarose percentages and well formats for your convenience A l
3. offers Contact Us For more information or technical assistance call write fax or email Additional international offices are listed on our website www invitrogen com Corporate Headquarters Japanese Headquarters European Headquarters Invitrogen Corporation Invitrogen Japan Invitrogen Ltd 5791 Van Allen Way LOOP X Bldg 6F Inchinnan Business Park Carlsbad CA 92008 USA 3 9 15 Kaigan 3 Fountain Drive Tel 1 760 603 7200 Minato ku Tokyo 108 0022 Paisley PA4 ORF UK Tel Toll Free 1 800 955 6288 Tel 81 3 5730 6509 Tel 44 0 141 814 6100 Fax 1 760 602 6500 Fax 81 3 5730 6519 Tech Fax 44 0 141 814 6117 E mail E mail E mail tech supportGinvitrogen com jpinfo invitrogen com eurotech invitrogen com MSDS MSDSs Material Safety Data Sheets are available on our website at www invitrogen com msds Certificate of Analysis The Certificate of Analysis CofA provides detailed quality control information for each product The CofA is available on our website at www invitrogen com cofa and is searchable by product lot number which is printed on each box Continued on next page 10 Technical Support Continued Limited Warranty 11 Invitrogen a part of Life Technologies Corporation is committed to providing our customers with high quality goods and services Our goal is to ensure that every customer is 100 satisfied with our products and our service If you shou
4. arge variety of DNA ladders is available from Invitrogen for sizing DNA For details on these products visit our website at www invitrogen com or contact Technical Support page 10 vii Overview Introduction System Overview Binding Buffer Introduction The PureLink PCR Micro Kit is designed for rapid and efficient purification of DNA from PCR products ranging in size from 125 bp 12 3 kb This kit allows you to isolate and purify high concentrations of DNA from PCR products in low elution volumes 10 pl Using the PureLink PCR Micro Kit gt 90 of dsDNA ssDNA primer dimmers less than 50 bp as well as dNTPs enzymes and salts are removed from your PCR products in approximately 6 minutes This manual provides a protocol for purifying high concentrations of DNA from PCR products in low elution volumes Binding Buffer B2 containing isopropanol is mixed with your PCR product in a 4 1 ratio This PCR product Binding Buffer mixture is added to the PureLink Micro Kit Column with a Collection Tube and is then centrifuged allowing the DNA to bind to the silica membrane of the column Impurities are subsequently removed from the silica membrane by the addition of Wash Buffer W1 containing ethanol The purified DNA is then eluted into the Elution Tube using a low salt Elution Buffer E1 The purified DNA is suitable for use in a wide variety of downstream applications next page The PureLink
5. brane and remove any residual Wash Buffer with ethanol Discard the flow through and the Collection Tube Reinsert the PureLink Micro Kit Column into an Elution Tube Add 10 pl Elution Buffer E1 10 mM Tris HCl pH 8 5 to the center of the PureLink Micro Kit Column Incubate for 1 minute at room temperature Centrifuge at 14 000 x g for 1 minute to collect the purified DNA into the Elution Tube Remove and discard the PureLink Micro Kit Column The Elution Tube now contains your purified DNA The recovered elution volume is 9 10 ul For long term storage store the purified DNA at 20 C For immediate use proceed to Analyzing DNA Yield and Quality next page or proceed to the downstream application of your choice Analyzing DNA Yield and Quality DNA Yield Primer Removal After product purification using the PureLink PCR Micro Kit the yield of purified DNA can be estimated by agarose gel electrophoresis or Quant iT DNA Assay Kits see page 6 Agarose Gel Electrophoresis To estimate the yield perform agarose gel electrophoresis of the purified PCR product and known quantities of DNA fragment of the same size Compare the band intensity of the purified PCR product with the standard DNA fragments Quant iT DNA Assay Kits The Quant iT DNA Assay Kits provide a rapid sensitive and specific method for dsDNA quantitation with minimal interference from RNA protein ssDNA primers or other com
6. e 2009 Life Technologies Corporation All rights reserved For research use only Not intended for any animal or human therapeutic or diagnostic use WAVE is a registered trademark of Transgenomic Inc invitrogen Corporate Headquarters Invitrogen Corporation 5791 Van Allen Way Carlsbad CA 92008 T 1 760 603 7200 F 1 760 602 6500 E tech_support invitrogen com For country specific contact information visit our web site at www invitrogen com
7. invitrogen PureLink PCR Micro Kit For rapidly purifying and concentrating DNA from PCR reactions in low elution volumes Catalog nos K310050 K310250 Rev Date 29 July 2009 Part no 100003661 IMPORTANT MAN0000408 New pre spin step added to protocol Table of Contents Experienced Users Procedure rrmmnernrsevrvrsrererraversaverersevesseversasersrseverseverssseressaverssser v Kit Contents and Storage msc see diia vi Accessory Products iii litio vii TNtrO UCR ERR ERR d ERO IER 1 OVerviem rarse m ee bra 1 TEE EE 4 Purification Procedure s cisscssssipssisesdsssnssesacassuscestcbanssedidissessecassenssessddssastocacsoians 4 Analyzing DNA Yield and Quality sse 7 Troubleshooting tette tete nere t ricetta tre 9 Technical Support EE 10 iii iv Experienced Users Procedure Introductions This quick reference page is provided for experienced users of the PureLink PCR Micro Kit If you are a first time user of this kit refer to the details provided in this manual Purification Before starting prepare Wash Buffer W1 with 100 ethanol Procedure and Binding Buffer B2 with 100 isopropanol page 5 Binding Washing and Elution of DNA Follow the steps below to bind wash and elute the DNA from your PCR product 1 Before using the PureLink Micro Kit Column centrifuge the column with collection tube for 1 minute at 10 000 x g 2 Add 4 vol
8. ld have any questions or concerns about an Invitrogen product or service contact our Technical Support Representatives All Invitrogen products are warranted to perform according to specifications stated on the certificate of analysis The Company will replace free of charge any product that does not meet those specifications This warranty limits the Company s liability to only the price of the product No warranty is granted for products beyond their listed expiration date No warranty is applicable unless all product components are stored in accordance with instructions The Company reserves the right to select the method s used to analyze a product unless the Company agrees to a specified method in writing prior to acceptance of the order Invitrogen makes every effort to ensure the accuracy of its publications but realizes that the occasional typographical or other error is inevitable Therefore the Company makes no warranty of any kind regarding the contents of any publications or documentation If you discover an error in any of our publications please report it to our Technical Support Representatives Life Technologies Corporation shall have no responsibility or liability for any special incidental indirect or consequential loss or damage whatsoever The above limited warranty is sole and exclusive No other warranty is made whether expressed or implied including any warranty of merchantability or fitness for a particular purpos
9. mon contaminants that affect UV absorbance The kit contains a state of the art quantitation reagent pre diluted standards for standard curve and a ready to use buffer The assay is performed in a microtiter plate format and is designed for reading in standard fluorescent microplate readers Follow manufacturer s recommendations to perform the assay The efficiency of primer removal can be estimated by agarose gel electrophoresis as described in the examples shown on the next page The WAVE System is an ideal method to estimate the efficiency of primer removal The WAVE System is an automated DHPLC denatured high performance liquid chromatography system Analyzing DNA Yield and Quality Continued Expected Results An example of efficient primer removal using the PureLink PCR Micro Kit is shown below Five micrograms 5 ug of Low DNA Mass Ladder page vii was mixed with an excess of a 37 mer primer and Binding Buffer with 100 isopropanol The mixture was purified using with the PureLink PCR Micro Kit as described in the manual The Binding Buffer DNA ladder primer mixture was analyzed prior to purification Lane 2 and after purification Lanes 4 5 using agarose gel electrophoresis Lane 1 is a 100 bp DNA Ladder page vii and Lane 3 is the 37 mer primer only 1 2 3 4 5 100 bp 37 mer primer Continued on next page Troubleshooting Problem Cause Soluti
10. on Low DNA yield PCR conditions Check amplicon on gel to verify the PCR not optimized product prior to purification Incorrect binding For efficient DNA binding always mix conditions 1 volume of PCR reaction with 4 volumes of Binding Buffer B2 containing isopropanol Be sure to add 100 isopropanol to the Binding Buffer as described on page 5 Ethanol not Be sure to add 100 ethanol to the Wash added to Wash Buffer W1 page 5 before use Buffer Membrane not Perform pre spin of PureLink Micro Kit seated properly Column before adding sample in column Incorrect elution Be sure to add Elution Buffer E1 to the conditions center of the column and perform incubation for I minute with Elution Buffer before centrifugation Inhibition of Presence of Traces of ethanol from the Wash Buffer downstream ethanol in can inhibit downstream enzymatic enzymatic purified DNA reactions reactions To remove the Wash Buffer discard the Wash Buffer flow through and centrifuge the spin column again at 14 000 x g for 1 minute to completely dry the column Technical Support Web Resources Visit the Invitrogen website at www invitrogen com for e Technical resources including manuals vector maps and sequences application notes MSDSs FAQs formulations citations handbooks etc e Complete technical support contact information e Access to the Invitrogen Online Catalog e Additional product information and special
11. r PCR products using the PureLink PCR Micro Kit Add appropriate Binding Spin PureLink PCR Spin Column Buffer with isopropanol in a Collection Tube briefly to the PCR products Apply sample to PureLink PCR Spin Column in a Collection Tube Wash column with Wash Buffer Elute DNA into PureLink Elution Tube i d ca y emi Methods Purification Procedure Introduction Materials Needed Important This purification procedure is designed to purify up to 5 ug dsDNA using a centrifuge in a total time of 6 minutes You will need the following items e 100 isopropanol e 100 ethanol e Microcentrifuge capable of centrifuging gt 14 000 x g Contents supplied with the kit e Binding Buffer B2 e Wash Buffer W1 e Elution Buffer E1 e PureLink Micro Kit Columns with Collection Tube e PureLink Elution Tubes e The Binding Buffer B2 contains guanidine hydrochloride This chemical is harmful when in contact with the skin or when it is inhaled or ingested e Do not add bleach or acidic solutions directly to solutions or sample preparation waste that contains guanidinium hydrochloride as reactive compounds and toxic gases are formed e The Wash Buffer W1 contains ethanol and the Binding Buffer B2 contains isopropanol Solutions containing ethanol or isopropanol are considered flammable Use appropriate precautions when using these chemicals For your protection always wear a labo
12. ratory coat gloves and safety glasses when handling these chemicals Dispose of the buffers and chemicals in appropriate waste containers Prior to using the PureLink Micro Kit Column spin the column in the collection tube briefly at 10 000 x g to ensure that the silica membrane is settled at the bottom of the column Purification Procedure Continued Preparing Binding Buffer with Isopropanol Preparing Wash Buffer with Ethanol EN Y Og ECO Before beginning prepare the Binding Buffer B2 with 100 isopropanol as follows For K310050 50 preps 1 Add 10 ml 100 isopropanol to the Binding Buffer 2 Check the box on the Binding Buffer label to indicate that isopropanol was added 3 Store the Binding Buffer with isopropanol at room temperature For K310250 250 preps 1 Add 48 ml 100 isopropanol to the Binding Buffer 2 Check the box on the Binding Buffer label to indicate that isopropanol was added 3 Store the Binding Buffer with isopropanol at room temperature Before beginning prepare the Wash Buffer W1 with 100 ethanol as follows For K310050 50 preps 1 Add 32 ml 100 ethanol to the Wash Buffer 2 Checkthe box on the Wash Buffer label to indicate that ethanol was added 3 Store the Wash Buffer with ethanol at room temperature For K310250 250 preps 1 Add 160 ml 10096 ethanol to the Wash Buffer 2 Checkthe box on the Wash Buffer label to indicate tha
13. t ethanol was added 3 Store the Wash Buffer with ethanol at room temperature Follow the recommendations below to obtain the best results e Use the recommended PCR volume of 50 100 ul e Save an aliquot of PCR products before purification to verify and check amplicon on the gel e Perform all centrifugation steps at room temperature e Pipet the Elution Buffer in the center of the column and perform a 1 minute incubation Continued on next page Purification Procedure Continued Binding Washing and Eluting DNA Follow the steps below to bind wash and elute the DNA from your PCR product 1 10 Before using the PureLink Micro Kit Column centrifuge the column with collection tube for 1 minute at 10 000 x g Add 4 volumes of Binding Buffer B2 with isopropanol to 1 volume of your PCR product e g add 200 ul Binding Buffer B2 with isopropanol to 50 pl PCR product Vortex or invert tube repeatedly to mix thoroughly then transfer the entire PCR product with Binding Buffer to a PureLink Micro Kit Column with a Collection Tube Centrifuge at 10 000 x g for 1 minute at room temperature Add 650 pl Wash Buffer W1 with ethanol to your sample in the PureLink Micro Kit Column Centrifuge at 10 000 x g for 1 minute at room temperature Discard the flow through and reinsert the PureLink Micro Kit Column into the Collection Tube Centrifuge at 14 000 x g for 1 minute to dry the silica mem
14. umes of Binding Buffer B2 with isopropanol to 1 volume of your PCR product e g add 200 ul Binding Buffer B2 with isopropanol to 50 ul PCR product 3 Vortex or invert tube repeatedly to mix thoroughly then transfer the entire PCR product with Binding Buffer to a PureLink Micro Kit Column with a Collection Tube 4 Centrifuge at 10 000 x g for 1 minute at room temperature 5 Add 650 ul Wash Buffer W1 with ethanol to your sample in the PureLink Micro Kit Column 6 Centrifuge at 10 000 x g for 1 minute at room temperature Discard the flow through and reinsert the PureLink Micro Kit Column into the Collection Tube 7 Centrifuge at 14 000 x g for 1 minute to dry the silica membrane and remove any residual Wash Buffer with ethanol Discard the flow through and the Collection Tube Reinsert the PureLink Micro Kit Column into an Elution Tube 8 Add 10 ul Elution Buffer E1 10 mM Tris HCl pH 8 5 to the center of the PureLink Micro Kit Column 9 Incubate for 1 minute at room temperature 10 Centrifuge at 14 000 x g for 1 minute to collect the purified DNA into the Elution Tube Remove and discard the PureLink Micro Kit Column The Elution Tube now contains your purified DNA The recovered elution volume is 9 10 ul For long term storage store the purified DNA at 20 C Kit Contents and Storage Shipping and Storage Kit Contents vi All contents of the PureLink PCR Micro Kits are shipped at
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