Sample & Assay Technologies DNeasy® mericon® Food
Contents
1. The optimized protocols for the DNeasy mericon Food Kit use CTAB in combination with Proteinase K to first digest compact tissue and to subsequently precipitate proteins with simultaneous precipitation of other cellular and food derived inhibitors Inhibitors are precipitated by centrifugation while the extracted DNA remains in solution In the subsequent chloroform extraction any remaining CTAB protein CTAB debris or CTAB polysaccharide complex not precipitated is removed along with other lipophilic inhibitors into the organic phase Only the aqueous phase containing the DNA and significantly depleted inhibitors is processed further This phase is mixed with binding buffer to adjust binding conditions and applied to the QlAquick Spin Columns The DNA obtained is ready for use in a downstream mericon real time PCR assay 5 DNeasy mericon Food Handbook 09 2010 7 Description of protocols The small fragment protocols are designed for extraction of total DNA from highly processed food material They have optimized binding conditions for the column purification and are recommended for strongly processed foods where DNA has been subjected to extensive thermal treatments e g cooking pasteurization etc high pressure irradiation pH changes or drying and is therefore highly fragmented down to 100 200 base pairs These protocols offer more stringent column binding conditions for small DNA fragments and have been successfully used fo2. In a globalized food market with increasing demand for food research and monitoring there is a need for streamlined testing solutions that are sensitive accurate and easy to use with a variety of starting materials The mericon food testing portfolio is a complete system of sample preparation and assay kits that meet the demands listed above Based on detection by real time polymerase chain reaction PCR mericon sample preparation kits and PCR Assays enable fast and reliable detection of a broad range of pathogens genetically modified organisms allergens and plant and animal matter in food animal feed or pharmaceutical products The DNeasy mericon Food Kit is designed for rapid up to 30 extractions in 2 5 hours purification of DNA from a variety of raw and processed food matrices while minimizing the carryover of PCR inhibitors inherent to complex food samples DNeasy mericon purified DNA is ready for use in a real time PCR using one of the mericon PCR Assays Principle and procedure The DNeasy mericon Food Kit uses modified cetyltrimethylammonium bromide CTAB extraction The nonionic detergent CTAB is widely used for efficient extraction of total nucleic acids from a wide range of tissue types Depending on the salt conditions CTAB may complex with nucleic acids low salt conditions or complex with inhibitors such as polysaccharides proteins and plant metabolites high salt conditions as found in the Food Lysis Buffer
3. 100 as indicated on the bottle to obtain a working solution Procedure 1 Place 2 g of homogenized food sample in a 50 ml centrifuge tube add 10 ml Food Lysis Buffer and 25 pl Proteinase K solution Vortex briefly to ensure complete distribution and moistening of the sample material Note For samples that swell greatly e g starches use double the amount of Food Lysis Buffer 20 ml to ensure that sufficient buffer solution covers the sample material 2 Incubate for 30 min at 60 C with constant shaking To enhance inhibitor precipitation cool the sample to room temperature 15 25 C on ice after incubation 3 Centrifuge for 5 min at 2500 x g Note The volume of supernatant strongly depends on the nature of the applied starting material and the amount of precipitated CTAB inhibitor complexes A range of 2 ml swelling foods e g homogenized cornflakes to 7 ml non swelling homogenized foods e g ketchup can be expected after centrifugation Make sure not carry over any precipitate from the bottom of the tube into the subsequent protocol steps 18 DNeasy mericon Food Handbook 09 2010 4 Pipet 500 pl chloroform into a 2 ml microcentrifuge tube Note Chloroform is a hazardous substance Always pipet chloroform in a fume hood Note As an organic solvent chloroform may leak from the pipet tip when transferred from one tube to another This can be avoided by calibrating the pipet tip to the solvent by repeatedly pi
4. 50 ml tubes Centrifuge with rotor for 2 ml tubes 200 mg starting sample Thermomixer for 2 ml tubes Shaking water bath at 60 C Shaking water bath at 60 C uu DNeasy mericon Food Handbook 09 2010 9 Equipment and Reagents to Be Supplied by User When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information consult the appropriate material safety data sheets MSDSs available from the product supplier Standard protocol and small fragment protocol 2 g E Homogenizer see Important Notes page 11 B Vortexer M Ethanol 96 100 E Chloroform M Centrifuge tubes 50 ml M Microcentrifuge tubes 1 5 ml or 2 ml Hi Centrifuge with rotor for 50 ml tubes M Microcentrifuge with rotor for 1 5 ml or 2 ml tubes capable of attaining 17 900 xg Hi Shaking incubator or shaking water bath capable of attaining 60 C Wi Pipets and pipet tips Standard protocol and small fragment protocol 200 mg Homogenizer see Important Notes page 11 Vortexer Ethanol 96 100 Chloroform Microcentrifuge tubes 2 ml Microcentrifuge with rotor for 2 ml tubes capable of attaining 17 900 x g Thermomixer for 2 ml tubes or shaking water bath capable of attaining 60 C Pipets and pipet tips Do not use denatured alcohol which contains other substances such as methanol or methylethylketone A ta 10 DNeasy mericon Food Handbook 09 2010 Important
5. Master Mix QuantiTect Nucleic Acid Dilution Buffer RNase free water mericon L pneumophila For 24 reactions mericon L 290093 Kit 24 pneumophila Assay Internal Control Positive Control Multiplex PCR Master Mix QuantiTect Nucleic Acid Dilution Buffer RNase free water mericon Shigella spp Kit For 24 reactions mericon Shigella spp 290103 24 Assay Internal Control Positive Control Multiplex PCR Master Mix QuantiTect Nucleic Acid Dilution Buffer RNase free water mericon Y enterocolitica For 24 reactions mericon Y 290113 Kit 24 enterocolitica Assay Internal Control Positive Control Multiplex PCR Master Mix QuantiTect Nucleic Acid Dilution Buffer RNase free water Larger kit sizes available please inquire 28 DNeasy mericon Food Handbook 09 2010 Product Related products Contents mericon GMO Detection Assays mericon Screen 35S Kit 24 mericon Screen Nos Kit 24 mericon RR Soy 24 For 24 reactions mericon Screen 35S Assay Internal Control Positive Control mericon Master Mix QuantiTect Nucleic Acid Dilution Buffer RNase free water For 24 reactions mericon Screen Nos Assay Internal Control Positive Control mericon Master Mix QuantiTect Nucleic Acid Dilution Buffer RNase free water For 24 reactions mericon RR Soy Assay Internal Control Positive Control mericon Master Mix QuantiTect Nucleic Acid Dilution Buffer RNase free water mericon Animal and P
6. Notes Homogenization Proper disruption of sample material in the protocols is not only important to facilitate food lysis and liberation of DNA but is also crucial to guarantee a homogeneous starting material representative of the whole food product In this context anticipated sensitivity and the amount of sample must be considered The higher the sensitivity requirements detection of trace amounts of food DNA e g allergens or genetically modified organisms GMOs or the more heterogeneous a food product is e g roughly chopped meats in a sausage the greater the amount of sample material required for homogenization in order to allow transfer of an overall representative sample into the procedure In addition to sample size sample type is also a deciding factor for the homogenization procedure Both aspects determine the homogenization device best suited for efficient disruption In order to select the best homogenization device it should be determined whether the food is soft hard or extremely hard Several options are available for each type of food see below Soft samples e g whole fruits in fruit jams or vegetables Hi Small amount of starting sample Small knife mill QIAGEN TissueRuptor hand blender E Large amount of starting sample Large knife mill Solid hard samples e g salami or frozen foods Hi Small amount of starting sample Small knife mill QIAGEN TissueLyser LT QIAGEN TissueLyser Il mortar
7. 00 988 0325 Denmark Orders 80 885945 Fax 80 885944 Technical 80 885942 Finland Orders 0800 914416 Fax 0800 914415 Technical 0800 914413 France Orders 01 60 920 926 Fax 01 60 920 925 Technical 01 60 920 930 Offers 01 60 920 928 Germany Orders 02103 29 12000 Fax 02103 29 22000 Technical 02103 29 12400 Hong Kong Orders 800 933 965 Fax 800 930 439 Technical 800 930 425 Ireland Orders 1800 555 049 Fax 1800 555 048 Technical 1800 555 061 Italy Orders 800 789 544 Fax 02 334304 826 Technical 800 787980 Japan Telephone 03 6890 7300 Fax 03 5547 0818 Technical 03 6890 7300 Korea South Orders 080 000 7146 Fax 02 2626 5703 Technical 080 000 7145 Luxembourg Orders 8002 2076 Fax 8002 2073 Technical 8002 2067 Mexico Orders 01 800 7742 639 Fax 01 800 1122 330 Technical 01 800 7742 436 The Netherlands Orders 0800 0229592 Fax 0800 0229593 Technical 0800 0229602 Norway Orders 800 18859 Fax 800 18817 Technical 800 18712 Singapore Orders 1800 742 4362 Fax 65 6854 8184 Technical 1800 742 4368 Spain Orders 91 630 7050 Fax 91 630 5145 Technical 91 630 7050 Sweden Orders 020 790282 Fax 020 790582 Technical 020 798328 Switzerland Orders 055 254 22 11 Fax 055 254 22 13 Technical 055 254 22 12 e Q e e UK Orders 01293 422 911 Fax 01293 422 922 Technical 01293 422 999 0 OS USA Orders 800 426 8157 Fax 800 718 2056 Technical 800 DNA PREP 800 362 7737 QI
8. AGEN Sample amp Assay Technologies
9. Buffer PB into a fresh 2 ml microcentrifuge tube add 350 pl of the upper aqueous phase from step 6 and mix thoroughly by vortexing 8 Pipet the solution from step 7 into the QlAquick spin column placed in a 2 ml collection tube Centrifuge at 17 900 x g for 1 min and discard the flow through Reuse the collection tube in step 9 9 Add 500 ul Buffer AW2 to the QlAquick spin column centrifuge at 17 900 x g for 1 min and discard the flow through Reuse the collection tube and centrifuge again at 17 900 x g for 1 min to dry the membrane Note Ensure that ethanol is added to Buffer AW2 See Things to do before starting page 13 Note Residual ethanol from Buffer AW2 will not be completely removed unless the flow through is discarded before the additional centrifugation 10 Transfer the QlAquick spin column to a 1 5 ml or 2 ml microcentrifuge tube not supplied and pipet 150 ul Buffer EB directly onto the QlAquick membrane Incubate for 1 min at room temperature 15 25C and then centrifuge at 17 900 x g for 1 min to elute 14 DNeasy mericon Food Handbook 09 2010 Protocol Standard Protocol 200 mg This protocol is designed for the extraction of total DNA from a small scale 200 mg sample of raw or processed food material Important points before starting W All centrifugation steps are carried out at room temperature 15 25 C in a microcentrifuge Hi Vortexing should be performed by pulse vortexing for 5 10 s E Thi
10. Insufficient or excess Optimize the amount of DNA used in the DNA used in downstream application if necessary downstream Downstream applications can be adversely application affected by insufficient or excess DNA DNeasy mericon Food Handbook 09 2010 25 References QIAGEN maintains a large up to date online database of scientific publications utilizing QIAGEN products Comprehensive search options allow you to find the articles you need either by a simple keyword search or by specifying the application research area title etc For a complete list of references visit the QIAGEN Reference Database online at www qiagen com RefDB search asp or contact QIAGEN Technical Services or your local distributor SSS S S lt gt XA 26 DNeasy mericon Food Handbook 09 2010 Ordering Information Product Contents mericon Sample Preparation Kits DNeasy mericon Food Kit 50 mericon DNA Bacteria Kit 100 mericon DNA Bacteria Plus Kit 50 mericon Assay Kits mericon Salmonella spp Kit 24 mericon L monocytogenes Kit 24 mericon Campylobacter spp Kit 24 mericon Campylobacter triple Kit 24 mericon VTEC stx1 2 Kit 24 50 QlAquick Spin Columns Proteinase K buffers Fast Lysis Buffer 50 Pathogen Lysis Tubes L Fast Lysis Buffer For 24 reactions mericon Salmonella Assay Internal Control Positive Control Multiplex PCR Master Mix QuantiTect Nucleic Acid Dilution Buffer RNase fr
11. September 2010 DNeasy mericon Food Handbook For extraction of total nucleic acids from a range of food sample types QIAGEN Sample amp Assay Technologies QIAGEN Sample and Assay Technologies QIAGEN is the leading provider of innovative sample and assay technologies enabling the isolation and detection of contents of any biological sample Our advanced high quality products and services ensure success from sample to result QIAGEN sets standards in Purification of DNA RNA and proteins Nucleic acid and protein assays microRNA research and RNAi Automation of sample and assay technologies Our mission is to enable you to achieve outstanding success and breakthroughs For more information visit www giagen com Contents Kit Contents 4 Storage 4 Product Use Limitations 4 Product Warranty and Satisfaction Guarantee 5 Quality Control 5 Technical Assistance 5 Safety Information 6 Introduction 7 Principle and procedure 7 Description of protocols 8 Equipment and Reagents to Be Supplied by User 10 Important Notes 11 Homogenization 11 Disruption using the QIAGEN TissueRuptor TissueLyser LT systems 12 Protocols NW Standard Protocol 2 g 13 NW Standard Protocol 200 mg 15 E Small Fragment Protocol 2 g 18 m Small Fragment Protocol 200 mg 20 Troubleshooting Guide 23 References 26 Ordering Information 27 DNeasy mericon Food Handbook 09 2010 3 Kit Contents DNeasy mericon Food Ki
12. This protocol is designed for the extraction of total DNA from a large scale 2 g sample of raw or processed food material Important points before starting W All centrifugation steps should be carried out at room temperature 15 25 C in a microcentrifuge MH Vortexing should be performed by pulse vortexing for 5 10 s Things to do before starting E Homogenize the food sample For information on disruption procedures and suitable disruption devices see Important Notes page 11 E Buffer AW2 is supplied as a concentrate Before using for the first time add the appropriate amount of ethanol 96 100 as indicated on the bottle to obtain a working solution Procedure 1 Place 2 g homogenized food sample in a 50 ml centrifuge tube add 10 ml Food Lysis Buffer and 25 pl Proteinase K solution Vortex briefly to ensure complete distribution and moistening of the sample material Note For samples that swell greatly e g starches double the amount of Food Lysis Buffer 20 ml to ensure that sufficient buffer solution covers the sample material 2 Incubate for 30 min at 60 C with constant shaking To enhance inhibitor precipitation cool the sample to room temperature 15 25 C on ice after incubation 3 Centrifuge for 5 min at 2500 x g Note The volume of supernatant strongly depends on the nature of the applied starting material and the amount of precipitated CTAB inhibitor complexes A range of 2 ml swelling foods e g ho
13. and pestle E Large amount of starting sample Large knife mill Extremely solid hard samples e g roots or seeds Hi Small amount of starting sample Small impact mill QIAGEN TissueLyser LT QIAGEN TissueLyser II E Large amount of starting sample Large impact mill DNeasy mericon Food Handbook 09 2010 11 Disruption using the QIAGEN TissueRuptor TissueLyser LT systems Homogenization using the QIAGEN TissueRuptor or TissueLyser systems is best carried out in combination with freezing the sample in liquid nitrogen This ensures optimal homogenization even with difficult sample material Disruption using the TissueRuptor should be carried out without Food Lysis Buffer after freezing the sample in liquid nitrogen Alternatively fresh material such as fruits or vegetables can be directly disrupted in Food Lysis Buffer without using liquid nitrogen however this may cause shearing of high molecular weight DNA We recommend keeping the disruption time to a minimum to avoid shearing of genomic DNA With the TissueLyser LT fresh material can be directly disrupted in lysis buffer without the use of liquid nitrogen Alternatively fresh or frozen samples can also be disrupted without lysis buffer after freezing in liquid nitrogen We do not recommend disruption of frozen material in lysis buffer as this can result in low yields and degraded DNA SSS EEE 12 DNeasy mericon Food Handbook 09 2010 Protocol Standard Protocol 2 g
14. andard protocol This is because supernatants are pooled in step 5 to ensure that the same amount of sample is processed during DNA purification Be sure to prepare sufficient lysis tubes to be able to pool samples 3 4 tubes Things to do before starting M Homogenize the food sample For information on disruption procedures and suitable disruption devices see Important Notes page 11 E Buffer AW2 is supplied as a concentrate Before using for the first time add the appropriate amount of ethanol 96 100 as indicated on the bottle to obtain a working solution 20 DNeasy mericon Food Handbook 09 2010 Procedure 1 Place 200 mg homogenized food sample in a 2 ml microcentrifuge tube add 1 ml Food Lysis Buffer and 2 5 ul Proteinase K solution Vortex briefly to ensure complete distribution and moistening of the sample material Note To ensure that DNA yields are similar to those obtained using the standard protocol 2 g supernatants are pooled in step 5 Depending on the starting material the supernatant from the 1 ml lysis solution will be less than 700 ul Be sure to prepare sufficient lysis tubes in the range of 3 4 lysis tubes so that supernatant aliquots from several lysis tubes can be pooled to draw the 700 ul optimal for subsequent chloroform extraction Note For samples that swell greatly e g starches double the amount of Food Lysis Buffer 2 ml to ensure that a sufficient level of buffer solution covers the sam
15. d unless the flow through is discarded before the additional centrifugation 11 Transfer the QlAquick spin column to a 1 5 ml or 2 ml microcentrifuge tube not supplied and pipet 150 pl Buffer EB directly onto the QlAquick membrane Incubate for 1 min at room temperature 15 25C and then centrifuge at 17 900 x g for 1 min to elute PF lt lt lt lt gt AAZZAZZZAZAZAZAZAZAZAAAEAAAEEEZEAA DNeasy mericon Food Handbook 09 2010 17 Protocol Small Fragment Protocol 2 g This protocol is designed for the extraction of total DNA from a large scale 2 g sample of raw or processed food material It has optimized column binding conditions adjusted for maximal recovery of short DNA fragments It is recommended for strongly processed foods where DNA has been subjected to extensive thermal treatments e g cooking pasteurization etc high pressure irradiation pH changes or drying and is therefore highly fragmented down to 100 200 base pairs Important points before starting E All centrifugation steps should be carried out at room temperature 15 25 C in a microcentrifuge Hi Vortexing should be performed by pulse vortexing for 5 10 s Things to do before starting M Homogenize the food sample For information on disruption procedures and suitable disruption devices see Important Notes page 11 W Buffer AW2 is supplied as a concentrate Before using for the first time add the appropriate amount of ethanol 96
16. ded to the lysis reaction If necessary extend incubation time at 60 C for Proteinase K digest to 90 min and or increase the amount of Proteinase K to 50 ul c Buffer AW2 prepared Make sure that ethanol has been added to incorrectly Buffer AW2 before use see Things to do before starting page 13 or 18 depending on which protocol is being used A _ _ 24 DNeasy mericon Food Handbook 09 2010 Comments and suggestions d Incorrect binding Make sure that the correct amount of lysate has conditions been pipetted after the chloroform extraction and is mixed 1 1 standard protocol or 1 4 small fragment protocol with Buffer PB to adjust the binding conditions correctly e DNA is still bound to Increase the volume of Buffer EB to 200 ul and the membrane incubate on the column for 5 min at room temperature 15 25 C before centrifugation DNA does not perform well in downstream experiments a Inhibitor carryover A possible inhibitor carryover is sometimes although not necessarily identified by a colored eluate Dilute the sample at least 1 10 before PCR analysis b Ethanol carryover Ensure that a dry spin step is performed after washing with Buffer AW2 The flow through after the wash needs to be discarded before the dry spin to allow complete drying of the membrane c Salt carryover Ensure that Buffer AW2 has been used at room temperature 15 25 C d
17. ee water For 24 reactions mericon L monocytogenes Assay Internal Control Positive Control Multiplex PCR Master Mix QuantiTect Nucleic Acid Dilution Buffer RNase free water For 24 reactions mericon Campylobacter spp Assay Internal Control Positive Control Multiplex PCR Master Mix QuantiTect Nucleic Acid Dilution Buffer RNase free water For 24 reactions mericon Campylobacter triple Assay Internal Control Positive Control Multiplex PCR Master Mix QuantiTect Nucleic Acid Dilution Buffer RNase free water For 24 reactions mericon VTEC stx1 2 Assay Internal Control Positive Control Multiplex PCR Master Mix QuantiTect Nucleic Acid Dilution Buffer RNase free water Larger kit sizes available please inquire DNeasy mericon Food Handbook 09 2010 Cat no 69514 69525 69534 290013 290023 290033 290043 290053 27 Product Contents Cat no mericon C sakazakii Kit For 24 reactions mericon C sakazakii 290063 24 Assay Internal Control Positive Control Multiplex PCR Master Mix QuantiTect Nucleic Acid Dilution Buffer RNase free water mericon S aureus Kit For 24 reactions mericon S aureus 290073 24 Assay Internal Control Positive Control Multiplex PCR Master Mix QuantiTect Nucleic Acid Dilution Buffer RNase free water mericon Legionella spp For 24 reactions mericon Legionella 290083 Kit 24 spp Assoy Internal Control Positive Control Multiplex PCR
18. eneous solution 6 Transfer 700 pl of the clear supernatant pool to the microcentrifuge tube containing the chloroform Note The supernatant can be strongly colored Certain foods may form three phases after centrifugation If this happens go through the upper phase with the pipet and transfer only an aliquot of the clear middle phase If the upper phase has formed a semi solid film for example as observed with chocolate pierce the film with the pipet and transfer only an aliquot of the clear middle phase 7 Vortex the microcentrifuge tube from step 6 vigorously for 15 s and centrifuge at 14 000 x g for 15 min Note If the supernatant is not clear centrifuge again for 5 min 8 Pipet 350 pl Buffer PB into a fresh 2 ml microcentrifuge tube add 350 pl of the upper aqueous phase from step 7 and mix thoroughly by vortexing 9 Pipet the solution from step 8 into the QlAquick spin column placed in a 2 ml collection tube Centrifuge at 17 900 x g for 1 min and discard the flow through Reuse the collection tube in step 10 16 DNeasy mericon Food Handbook 09 2010 10 Add 500 pl Buffer AW2 to the QlAquick spin column centrifuge at 17 900 x g for 1 min and discard flow through Reuse the collection tube and centrifuge again at 17 900 x g for 1 min to dry the membrane Note Ensure that ethanol is added to Buffer AW2 See Things to do before starting page 13 Note Residual ethanol from Buffer AW2 will not be completely remove
19. ermine the suitability of the product for its particular use Should any product fail to perform satisfactorily due to any reason other than misuse QIAGEN will replace it free of charge or refund the purchase price We reserve the right to change alter or modify any product to enhance its performance and design If a QIAGEN product does not meet your expectations simply call your local Technical Service Department or distributor We will credit your account or exchange the product as you wish Separate conditions apply to QIAGEN scientific instruments service products and to products shipped on dry ice Please inquire for more information A copy of QIAGEN terms and conditions can be obtained on request and is also provided on the back of our invoices If you have questions about product specifications or performance please call QIAGEN Technical Services or your local distributor see back cover or visit www qiagen com Quality Control In accordance with QIAGEN s ISO certified Quality Management System each lot of DNeasy mericon Food Kit is tested against predetermined specifications to ensure consistent product quality Technical Assistance At QIAGEN we pride ourselves on the quality and availability of our technical support Our Technical Service Departments are staffed by experienced scientists with extensive practical and theoretical expertise in sample and assay technologies and the use of QIAGEN products If you have any q
20. k and safety phrases apply to components of the DNeasy mericon Food Kit Food Lysis Buffer Contains cetyltrimethylammonium bromide CTAB Dangerous for the environment Risk and safety phrases R52 53 Proteinase K Contains Proteinase K Sensitizer irritant Risk and safety phrases R36 37 38 42 43 S23 24 26 36 37 Buffer PB Contains guanidine hydrochloride isopropanol Harmful irritant flammable Risk and safety phrases R10 22 36 38 S13 23 26 36 37 39 46 24 hour emergency information Emergency medical information in English French and German can be obtained 24 hours a day from Poison Information Center Mainz Germany Tel 49 6131 19240 R10 Flammable R22 Harmful if swallowed R36 38 Irritating to eyes and skin R52 53 Harmful to aquatic organisms may cause long term adverse effects in the aquatic environment R36 37 38 Irritating to eyes respiratory system and skin R42 43 May cause sensitization by inhalation and skin contact S13 Keep away from food drink and animal feedingstuffs S23 Do not breathe vapor S24 Avoid contact with the skin 26 In case of contact with eyes rinse immediately with plenty of water and seek medical advice S36 37 Wear suitable protective clothing and gloves S36 37 39 Wear suitable protective clothing gloves and eye face protection S46 If swallowed seek medical advice immediately and show container or label 6 DNeasy mericon Food Handbook 09 2010 Introduction
21. lant Identification Assays mericon Pig Kit 24 mericon Pig Kit 24 For 24 reactions mericon Pig Assay Internal Control Positive Control mericon Master Mix QuantiTect Nucleic Acid Dilution Buffer RNase free water For 24 reactions mericon Pig Assay Internal Control Positive Control mericon Master Mix QuantiTect Nucleic Acid Dilution Buffer RNase free water Cat no 291013 291043 291113 292013 292013 For up to date licensing information and product specific disclaimers see the respective QIAGEN kit handbook or user manual QIAGEN kit handbooks and user manuals are available at www giagen com or can be requested from QIAGEN Technical Services or your local distributor Larger kit sizes available please inquire DNeasy mericon Food Handbook 09 2010 29 Notes 30 DNeasy mericon Food Handbook 09 2010 Trademarks QIAGEN QlAquick DNeasy mericon QuantiTect TissueRuptor QIAGEN Group Limited License Agreement Use of this product signifies the agreement of any purchaser or user of the DNeasy mericon Food Kit to the following terms 1 an Gr S9 The DNeasy mericon Food Kit may be used solely in accordance with the DNeasy mericon Food Handbook and for use with components contained in the Kit only QIAGEN grants no license under any of its intellectual property to use or incorporate the enclosed components of this Kit with any components not included within this Kit exce
22. letely removed unless the flow through is discarded before the additional centrifugation 12 Transfer the QlAquick spin column to a 1 5 ml or 2 ml microcentrifuge tube not supplied and pipet 100 pl Buffer EB directly onto the QlAquick membrane Incubate for 1 min at room temperature 15 25 C and then centrifuge at 17 900 x g for 1 min to elute 22 DNeasy mericon Food Handbook 09 2010 Troubleshooting Guide This troubleshooting guide may be helpful in solving problems that may arise For more information see also the Frequently Asked Questions page at our Technical Support Center www giagen com FAQ FAQList aspx The scientists in QIAGEN Technical Services are always happy to answer any questions you may have about either the information and protocols in this handbook or sample and assay technologies for contact information see back cover or visit www qiagen com Comments and suggestions Solid film formed on the lysis solution after incubation at 60 C and subsequent centrifugation Liberated food Continue with the protocol and pierce any top components are layer with the pipet Carefully draw the 700 pl deposited or aliquot from the clear middle phase making compacted on top of sure that the pipet tip is not blocked by food the reaction solution deposits after lysis No supernatant from which to draw the 700 pl aliquot after incubation at 60 C and subsequent centrifugation a Insufficient food Make sure that the food i
23. mogenized cornflakes to 7 ml non swelling homogenized foods e g ketchup can be expected after centrifugation Do not carry over any precipitate from the bottom of the tube into the subsequent protocol steps lau DNeasy mericon Food Handbook 09 2010 13 4 Pipet 500 pl chloroform into a 2 ml microcentrifuge tube Note Chloroform is a hazardous substance Always pipet chloroform in a fume hood Note As an organic solvent chloroform may leak from the pipet tip when transferred from one tube to another This can be avoided by calibrating the pipet tip to the solvent by repeatedly pipetting up and down before transferring a specific volume 5 Carefully transfer 700 pl of the clear supernatant from step 3 to the microcentrifuge tube containing the chloroform Be sure not to carry over material from the bottom phase which contains precipitated food debris Note The supernatant can be strongly colored Certain foods may also form three phases after centrifugation If this happens go through the upper phase with the pipet and transfer only 700 ul of the clear middle phase If the upper phase has formed a semi solid film for example as observed with chocolate pierce the film with the pipet and transfer only 700 ul of the clear middle phase 6 Vortex the microcentrifuge tube from step 5 vigorously for 15 s and centrifuge at 14 000 x g for 15 min Note If the supernatant is not clear centrifuge again for 5 min 7 Pipet 350 pl
24. petting up and down before transferring a specific volume 5 Carefully transfer 700 pl of the clear supernatant from step 3 to the microcentrifuge tube containing the chloroform Be sure not to carry over material from the bottom phase which contains precipitated food debris Note The supernatant can be strongly colored Certain foods may also form three phases after centrifugation If this happens go through the upper phase with the pipet and transfer 700 ul only of the clear middle phase If the upper phase has formed a semi solid film for example as observed with chocolate pierce the film with the pipet and transfer only 700 ul of the clear middle phase 6 Vortex the microcentrifuge tube from step 5 vigorously for 15 s and centrifuge at 14 000 x g for 15 min Note If the supernatant is not clear centrifuge again for 5 min 7 Pipet 1 ml Buffer PB into a fresh 2 ml microcentrifuge tube add 250 pl of the upper aqueous phase from step 6 and mix thoroughly by vortexing 8 Pipet 600 pl of the mixture from step 7 into the QlAquick spin column placed in a 2 ml collection tube Centrifuge at 17 900 x g for 1 min and discard the flow through Reuse the collection tube in step 9 9 Repeat step 8 with remaining sample and discard flow through Reuse the collection tube in step 10 10 Add 500 pl Buffer AW2 to the QlAquick spin column centrifuge at 17 900 x g for 1 min and the discard flow through Reuse the collection tube and cent
25. ple material 2 Incubate in a thermomixer for 30 min at 60 C with constant shaking 1000 rpm To enhance inhibitor precipitation cool the sample to room temperature 15 25 C on ice after incubation 3 Centrifuge for 5 min at 2500 x g Note The volume of supernatant strongly depends on the nature of the applied starting material and the amount of precipitated CTAB inhibitor complexes A range of 200 ul swelling foods e g homogenized cornflakes to 700 ul non swelling homogenized foods e g ketchup can be expected after centrifugation Make sure not carry over any precipitate from the bottom of the tube into the subsequent protocol steps 4 Pipet 500 pl chloroform into a 2 ml microcentrifuge tube Note Chloroform is a hazardous substance Always pipet chloroform in a fume hood Note As an organic solvent chloroform may leak from the pipet tip when transferred from one tube to another This can be avoided by calibrating the pipet tip to the solvent by repeatedly pipetting up and down before transferring a specific volume 5 Carefully draw the maximum volume of clear supernatant from each lysis tube replicate from step 3 without disturbing the inhibitor precipitate at the bottom at the tube Combine the supernatant aliquots in one microcentrifuge tube and mix by pipetting up and down several times to ensure a homogeneous solution DNeasy mericon Food Handbook 09 2010 21 6 Transfer 700 pl of the clear supernatant pool
26. pt as described in the DNeasy mericon Food Handbook and additional protocols available at www giagen com Other than expressly stated licenses QIAGEN makes no warranty that this Kit and or its use s do not infringe the rights of third parties This Kit and its components are licensed for one time use and may not be reused refurbished or resold QIAGEN specifically disclaims any other licenses expressed or implied other than those expressly stated The purchaser and user of the Kit agree not to take or permit anyone else to take any steps that could lead to or facilitate any acts prohibited above QIAGEN may enforce the prohibitions of this Limited License Agreement in any Court and shall recover all its investigative and Court costs including attorney fees in any action to enforce this Limited License Agreement or any of its intellectual property rights relating to the Kit and or its components For updated license terms see www giagen com 2010 QIAGEN all rights reserved www giagen com Australia Orders 1 800 243 800 Fax 03 9840 9888 Technical 1 800 243 066 Austria Orders 0800 28 10 10 Fax 0800 28 10 19 Technical 0800 28 10 11 Belgium Orders 0800 79612 Fax 0800 79611 Technical 0800 79556 Brazil Orders 0800 557779 Fax 55 11 5079 4001 Technical 0800 557779 Canada Orders 800 572 9613 Fax 800 713 5951 Technical 800 DNA PREP 800 362 7737 China Orders 86 21 3865 3865 Fax 86 21 3865 3965 Technical 8
27. r DNA extraction from foods such as flour and cornflakes For all other food types and raw materials the standard protocols should be used The standard protocols are designed for the extraction of total DNA from complex food samples for which a lower processing grade and thus less DNA fragmentation is expected We have successfully extracted DNA from foods such as ketchup and chocolate This handbook contains 2 standard and 2 small fragment protocols depending on the sample input size large scale 2 g or small scale 200 mgl Each protocol has scale specific adjustments Therefore sample and input size should be considered for technical prerequisites see Equipment and Reagents to Be Supplied by User page 10 However food samples can be very complex and the processing grade of a specific food is not always clear It is therefore not possible to predict which protocol will work best for each sample The flowchart on the following page may be used as a guide to which protocol is best suited for your sample AAA CE CC LC LECCE LLLI 8 DNeasy mericon Food Handbook 09 2010 Food processing grade DNA fragmentation grade DNA extraction from foods with high DNA extraction from foods with little processing for example or normal processing for example E Flour E Chocolate E Cornflakes E Ketchup Small Fragment Protocol Standard Protocol Availability of lysis equipment Centrifuge with rotor for
28. rifuge again at 17 900 x g for 1 min to dry the membrane Note Ensure that ethanol is added to Buffer AW2 See Things to do before starting page 18 Note Residual ethanol from buffer AW2 will not be completely removed unless the flow through is discarded before the additional centrifugation 11 Transfer the QlAquick spin column to a 1 5 ml or 2 ml microcentrifuge tube not supplied and pipet 100 ul Buffer EB directly onto the QlAquick membrane Incubate for 1 min at room temperature 15 25 C and then centrifuge at 17 900 x g for 1 min to elute DNeasy mericon Food Handbook 09 2010 19 Protocol Small Fragment Protocol 200 mg This protocol is designed for the extraction of total DNA from a small scale 200 mg sample of raw or processed food material Column binding conditions are optimized for a maximal recovery of short DNA fragments It is recommended for strongly processed foods where DNA has been subjected to extensive thermal treatments e g cooking pasteurization etc high pressure irradiation pH changes or drying and is therefore highly fragmented down to 100 200 base pairs Important points before starting E All centrifugation steps should be carried out at room temperature 15 25 C in a microcentrifuge Hi Vortexing should be performed by pulse vortexing for 5 10 s M This protocol uses a smaller starting food sample size However overall DNA yields should be similar to those obtained using the st
29. s completely disruption and homogenized before adding the Food Lysis subsequent swelling of Buffer food e g cornflakes b Strong swelling of Apply the same amount of sample but double already homogenized the amount of Food Lysis Buffer food e g starches DNeasy mericon Food Handbook 09 2010 23 Comments and suggestions QlAquick membrane is colored Food inhibitors carried After washing with Buffer AW2 perform an over from lysis inhibitor additional wash step using 500 ul ethanol precipitation and 96 100 Centrifuge for 2 min at 20 000 x y chloroform extraction to dry the membrane and continue with the are deposited on the protocol membrane In general inhibitors are one of the following M Retained by the membrane Membrane remains colored after washing and elution but DNA elution and quality are unaffected M Removed by additional ethanol wash step M Co eluted into DNA solution See DNA does not perform well in downstream experiments below DNA eluate is colored Inhibitor carryover See DNA does not erform well in downstream y experimer ts below Low DNA yield a Insufficient disruption Ensure that the starting material is completely disrupted See Disruption using the QIAGEN TissueRuptor TissueLyser LT systems pagel2 b Insufficient lysis Reduce the amount of starting material and or increase the amount of Food Lysis Buffer Check that the correct amount of Proteinase K has been ad
30. s protocol uses a smaller starting food sample size However overall DNA yields should be similar to those obtained using the standard protocol This is because supernatants are pooled in step 5 to ensure that the same amount of sample is processed during DNA purification Be sure to prepare sufficient lysis tubes to be able to pool samples 3 4 tubes Things to do before starting E Homogenize the food sample For information on disruption procedures and suitable disruption devices see Important Notes page 11 E Buffer AW2 is supplied as a concentrate Before using for the first time add the appropriate amount of ethanol 96 100 as indicated on the bottle to obtain a working solution Procedure 1 Place 200 mg homogenized food sample in a 2 ml microcentrifuge tube add 1 ml Food Lysis Buffer and 2 5 ul Proteinase K solution Vortex briefly to ensure complete distribution and moistening of the sample material Note To ensure that DNA yields are similar to those obtained using the standard protocol 2 g supernatants are pooled in step 5 Depending on the starting material the supernatant from the 1 ml lysis solution will be less than 700 pl Be sure to prepare sufficient lysis tubes in the range of 3 4 lysis tubes so that supernatant aliquots from several lysis tubes can be pooled to draw the 700 ul optimal for subsequent chloroform extraction Note For samples that swell greatly e g starches double the amount of Food L
31. t 50 Catalog no 69514 Number of preps 50 Food Lysis Buffer 4 x 200 ml Proteinase K 1 4 ml QlAquick Spin Columns 50 Buffer PB 2 x 30 ml Buffer AW2 concentrate 13 ml Buffer EB 15 ml Handbook 1 Storage DNeasy mericon Food Kit components should be stored dry at room temperature 15 25 C The DNeasy mericon Food Kit can be stored at 2 8 C but buffers should be redissolved at 37 C before use if precipitates are observed Ensure that all buffers and spin columns are at room temperature 15 25 C before use DNeasy mericon Food Kits contain a ready to use Proteinase K solution which is supplied in a specially formulated storage buffer Proteinase K is stable for at least 1 year after delivery when stored at room temperature For storage longer than one year or if ambient temperatures often exceed 25 C we suggest storing Proteinase K solution at 2 8 C DNeasy mericon Food Kit components are stable for 1 year under these conditions without showing reduction in performance and quality Product Use Limitations The DNeasy mericon Food Kit is intended for molecular biology applications in food animal feed and pharmaceutical product testing This product is not intended for the diagnosis prevention or treatment of a disease 4 DNeasy mericon Food Handbook 09 2010 Product Warranty and Satisfaction Guarantee QIAGEN guarantees the performance of all products in the manner described in our product literature The purchaser must det
32. to the microcentrifuge tube containing the chloroform Note The supernatant can be strongly colored Certain foods may also form three phases after centrifugation If this happens go through the upper phase with the pipet and transfer only an aliquot of the clear middle phase If the upper phase has formed a semi solid film for example as observed with chocolate pierce the film with the pipet and transfer only an aliquot of the clear middle phase 7 Vortex the microcentrifuge tube from step 6 vigorously for 15 s and centrifuge at 14 000 x g for 15 min Note If the supernatant is not clear centrifuge again for 5 min 8 Pipet 1 ml Buffer PB into a fresh 2 ml microcentrifuge tube add 250 pl of the upper aqueous phase from step 7 and mix thoroughly by vortexing 9 Pipet 600 pl of the mixture from step 8 into the QlAquick spin column placed in a 2 ml collection tube Centrifuge at 17 900 x g for 1 min and discard the flow through Reuse the collection tube in step 9 10 Repeat step 9 with remaining sample and discard flow through Reuse the collection tube in step 11 11 Add 500 pl Buffer AW2 to the QlAquick spin column centrifuge at 17 900 x g for 1 min and the discard flow through Reuse the collection tube and centrifuge again at 17 900 x g for 1 min to dry the membrane Note Ensure that ethanol is added to Buffer AW2 See Things to do before starting page 18 Note Residual ethanol from buffer AW2 will not be comp
33. uestions or experience any difficulties regarding the DNeasy mericon Food Kit or QIAGEN products in general please do not hesitate to contact us QIAGEN customers are a major source of information regarding advanced or specialized uses of our products This information is helpful to other scientists as well as to the researchers at QIAGEN We therefore encourage you to contact us if you have any suggestions about product performance or new applications and techniques For technical assistance and more information please see our Technical Support Center at www giagen com Support or call one of the QIAGEN Technical Service Departments or local distributors see back cover or visit www giagen com DNeasy mericon Food Handbook 09 2010 5 Safety Information When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information please consult the appropriate material safety data sheets MSDSs These are available online in convenient and compact PDF format at www giagen com Support MSDS aspx where you can find view and print the MSDS for each QIAGEN kit and kit component CAUTION Do not add bleach directly to the sample preparation waste Buffer PB contains guanidine hydrochloride which can form highly reactive compounds when combined with bleach In case liquid containing this buffer is spilt clean with suitable laboratory detergent and water The following ris
34. ysis Buffer 2 ml to ensure that sufficient buffer solution covers the sample material 2 Incubate in a thermomixer for 30 min at 60 C with constant shaking 1000 rpm To enhance inhibitor precipitation cool the sample to room temperature 15 25 C on ice after incubation DNeasy mericon Food Handbook 09 2010 15 3 Centrifuge for 5 min at 2500 x g Note The volume of supernatant strongly depends on the nature of the applied starting material and the amount of precipitated CTAB inhibitor complexes A range of 200 ul swelling foods e g homogenized cornflakes to 700 ul non swelling homogenized foods e g ketchup can be expected after centrifugation Make sure not to carry over any precipitate from the bottom of the tube into the subsequent protocol steps 4 Pipet 500 pl chloroform into a 2 ml microcentrifuge tube Note Chloroform is a hazardous substance Always pipet chloroform in a fume hood Note As an organic solvent chloroform may leak from the pipet tip when transferred from one tube to another This can be avoided by calibrating the pipet tip to the solvent by repeatedly pipetting up and down before transferring a specific volume 5 Carefully draw the maximum volume of clear supernatant from each lysis tube from step 3 without disturbing the inhibitor precipitate at the bottom at the tube Combine the supernatant aliquots in one microcentrifuge tube and mix by pipetting up and down several times to ensure a homog
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