Sample & Assay Technologies QIAamp® UCP
Contents
1. EB Ensure that Buffer APR Buffer APB1 Buffer APW1 and Buffer APW2 have been prepared according to the instructions on page 14 Procedure 1 Place 3 8 ml of fresh blood into a 15 ml centrifuge tube not provided Close the cap and centrifuge 5 000 x g for 10 min This step separates plasma from blood and microbial cells with one volume of fresh blood approximately separating into 50 cell fraction and 50 plasma fraction 2 Open the cap and carefully transfer the upper plasma layer plasma fraction into a sterile 50 ml centrifuge tube not provided Save the plasma later for step 11 Note Do not disturb the white blood cell layer when transferring the plasma layer 3 Add approximately 1 volume of Buffer APL1 to the remaining cell fraction from step 1 and resuspend the pellet by vigorous vortexing and pipetting for 30 s For instance add approximately 4 ml Buffer APL to 4 ml of the remaining cell fraction not total volume of blood provided in step 1 Buffer APL1 specifically lyses human blood cells while microbial cells stay intact 4 Close the tube and incubate the cell fraction buffer mixture for 10 min at room temperature 15 25 C 5 Centrifuge the cell fraction buffer mixture for 10 min 5 000 x g 6 Carefully discard the supernatant but save the microbial cell pellet 20 QlAamp UCP PurePathogen Blood Handbook 04 201 1 Note Do not vigorously shake the tube while discarding the supernatant A small fracti2. Apply 600 ul of Buffer APW1 to the QlAamp UCP Mini column Carefully remove and discard the extension tube and switch on the vacuum pump After all of Buffer APW1 has been drawn through the Mini column switch off the vacuum pump and release the pressure to O mbar To avoid cross contaminations be careful to not cross neighboring QlAamp UCP Mini Columns while extension tubes are removed Apply 750 ul of Buffer APW2 to the QlAamp UCP Mini column Leave the lid of the column open and switch on the vacuum pump After all of Buffer APW2 has been drawn through the Mini column switch off the vacuum pump and release the pressure to 0 mbar Apply 750 ul of ethanol 96 100 to the QlAamp UCP Mini column Leave the lid of the column open and switch on the vacuum pump After all of ethanol has been drawn through the spin column switch off the vacuum pump and release the pressure to 0 mbar Close the lid of the QlAamp UCP Mini column remove it from the vacuum manifold and discard the VacConnector Place the QlAamp UCP Mini column in a clean 2 ml collection tube and centrifuge at full speed 20 000 x g 14 000 rpm for 3 min Place the QlAamp UCP Mini Column into a new 2 ml collection tube open the lid and incubate the assembly at 56 C for 3 min to dry the membrane completely Place the QlAamp UCP Mini column in a clean 1 5 ml elution tube and discard the collection tube Carefully apply 150 pl of Buffer AVE to the center of the QlAamp Mini m
3. If the spilt liquid contains potentially infectious agents clean the affected area first with laboratory detergent and water and then with 1 v v sodium hypochlorite The following risk and safety phrases apply to components of QlAamp UCP PurePathogen Blood Kit Buffer APL2 and Buffer APB1 Contain guanidine thiocyanate harmful Risk and safety phrases R20 21 22 32 S13 26 36 37 39 46 Buffer APW1 Contains guanidine hydrochloride harmful irritant Risk and safety phrases R22 36 38 S13 26 36 46 Proteinase K Contains proteinase K sensitizer irritant Risk and safety phrases R36 37 38 42 43 S23 24 26 36 37 R20 21 22 Harmful by inhalation in contact with skin and if swallowed R22 Harmful if swallowed R32 Contact with acids liberates very toxic gas R36 37 38 Irritating to eyes respiratory system and skin R36 38 Irritating to eyes and skin R42 43 May cause sensitization by inhalation and skin contact 13 Keep away from food drink and animal feeding stuffs 523 Do not breathe vapor S24 Avoid contact with the skin S26 In case of contact with eyes rinse immediately with plenty of water and seek medical advice S36 Wear suitable protective clothing 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 QlAamp UCP PurePathogen Blood Handbook 04 2
4. between blood draw target nucleic acid Use the protocol for thawed and plasma blood preparation b Inefficient lysis of The human DNA gets discarded after lysis of the leukocytes leukocytes using Buffer APL Make sure that the supernatant gets discarded after 10 min incubation at room temperature Eluted nucleic acids do not perform well in downstream reactions a Little or no DNA in the See Little or no nucleic acids in the eluate eluate above for possible reasons for little or no DNA in the eluate Increase the amount of eluate added to the reaction if possible b Inappropriate elution Determine the maximum volume of eluate volume used suitable for your downstream reaction Reduce or increase the volume of eluate added to the reaction accordingly The elution volume can be adapted proportionally c Buffers not mixed Salt and ethanol components of wash Buffer thoroughly APW2 may have separated out after being left for a long period between runs Always mix buffers thoroughly before each run d High human DNA High amounts of human DNA in the eluate may background inhibit downstream reactions See High human DNA background above for possible reasons Decrease the amount of eluate added to the reaction if possible 28 QlAamp UCP PurePathogen Blood Handbook 04 201 1 e Buffers APW1 and APW2 used in the wrong order f Reduced sensitivity of downstream reaction g Residual ethanol in the eluate General handlin
5. M Centrifuge with rotor for 15 ml and 50 ml centrifuge tubes E Vacuum manifold e g QlAvac 24 Plus cat no 19413 M Vacuum Regulator cat no 19530 for easy monitoring of vacuum pressures and easy releasing of vacuum M Ethanol 96 100 E isopropanol 100 E Optional VacValves cat no 19408 E Optional QlAvac Connecting System cat no 19419 Do not use denatured alcohol which contains other substances such as methanol or methylethylketone QlAamp UCP PurePathogen Blood Handbook 04 2011 13 Important Notes Preparation of buffers and reagents Buffer APR Before use add 100 ul Reagent DX to one bottle of buffer APR If smaller amounts are needed before use transfer 1 5 ml of buffer APR into a sterile 2 ml vial and add 10 ul Reagent DX Mix well after adding Reagent DX After preparation the mixture is stable for 6 months at room temperature 15 25 C Buffer APB1 Before use add 40 ml isopropanol 100 to one bottle of buffer APB1 concentrate 20 ml to obtain 60 ml Buffer APB1 Mix well after adding isopropanol Buffer APW1 Before use add 8 ml ethanol 96 100 to 6 ml buffer APW1 concentrate to obtain 14 ml Buffer APW1 Mix well after adding ethanol Buffer APW2t Before use add 10 ml ethanol 96 100 to 4 ml buffer APW2 concentrate to obtain 14 ml Buffer APW2 Mix well after adding ethanol QlAvac 24 Plus The QlAvac 24 Plus is designed for fast and efficient vacuum processing of up to 24 Q
6. Orders 800 426 8157 Fax 800 718 2056 Technical 800 DNA PREP 800 362 7737 QIAGEN Sample amp Assay Technologies
7. 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 Appendix A General Remarks General handling Proper microbiological aseptic technique should always be used when purifying nucleic acids with the QlAamp UCP PurePathogen Blood Kit Hands and dust particles may carry bacteria and molds and are the most common sources of contamination Always wear latex or vinyl gloves while handling reagents and consumables Change gloves frequently and keep tubes closed whenever possible Keep purified nucleic acids on ice when aliquots are pipetted for downstream applications Disposable plasticware The use of sterile disposable polypropylene tubes is recommended throughout the procedure These tubes are generally free of contaminating nucleic acids as well as nuclease and do not require pretreatment Sas QlAamp UCP PurePathogen Blood Handbook 04 2011 31 Ordering Information Product Contents Cat no QlAamp UCP For 10 preps QlAamp UCP Mini 50112 PurePathogen Kit 10 Columns Pathogen Lysis Tubes L Tube Extenders 20 ml Proteinase K Buffers VacConnectors and Collection Tubes 1 5 ml and 2 ml Accessories QlAvac 24 Plus Vacuum manifold for processing 1 24 19413 spin columns includes QlAvac 24 Plus Vacuum Manifold Luer Plugs Quick Couplings VacValves 24 24 valves for use
8. column Leave the lid of the column open and switch on the vacuum pump After all of Buffer APW2 has been drawn through the Mini column switch off the vacuum pump and release the pressure to 0 mbar Apply 750 ul of ethanol 96 100 to the QlAamp UCP Mini column Leave the lid of the column open and switch on the vacuum pump After all of ethanol has been drawn through the spin column switch off the vacuum pump and release the pressure to 0 mbar Close the lid of the QlAamp UCP Mini column remove it from the vacuum manifold and discard the VacConnector Place the QlAamp UCP Mini column in a clean 2 ml collection tube and centrifuge at full speed 20 000 x g 14 000 rpm for 3 min Place the QlAamp UCP Mini column into a new 2 ml collection tube open the lid and incubate the assembly at 56 C for 3 min to dry the membrane completely Place the QlAamp UCP Mini column in a clean 1 5 ml elution tube and discard the collection tube Carefully apply 150 pl of Buffer AVE to the center of the QlAamp Mini membrane Close the lid and incubate at room temperature for 3 min IMPORTANT Ensure that the elution buffer is equilibrated to room temperature Elution volume is flexible and can be adapted according to the requirements of downstream applications The recovered eluate volume will be up to 5 ul less than the elution volume applied onto the column If elution is done in small volumes 100 ul the elution buffer has to be dispensed onto the cent
9. 01 1 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 Quality Control In accordance with QIAGEN s ISO certified Quality Management System each lot of QlAamp UCP PurePathogen Blood Kit is tested against predetermined specifications to ensure consistent product quality QlAamp UCP PurePathogen Blood Handbook 04 2011 7 Introduction The QlAamp UCP PurePathogen Blood Kit uses ultraclean production UCP technology The QlAamp UCP PurePathogen Blood Kit enables purification of nucleic acids from bacteria and fungi which are difficult to lyse as well as viral and free circulating nucleic acids from fresh or frozen EDTA treated whole blood samples with depletion of human genomic DNA The eluted nucleic acids including viral bacterial and fungal nucleic acids are ready for use in common downstream reactions or storage at 20 to 30 C The purified nucleic acids have no detectable contaminating proteins nucleases and other impurities Principle and procedure For maximal sensitivity in detection of pathogens from whole blood samples large blood volumes are needed to purify nucleic acids because pathogen titers are often very low Eluates from whole blood purification usually contain high concentrations of human DNA that could inhibit downstream reactions The QlAamp UCP PurePathogen Blood
10. 1 1 17 Figure 2 Setting up the QlAvac 24 Plus with QlAamp UCP Mini columns using VacValves VacConnectors and Tube Extenders 1 QlAvac 24 Plus vacuum manifold 4 VacConnector 2 Luer slot of the QlAvac 24 Plus closed with 5 QlAamp UCP Mini column luer plug 3 VacValve 6 Tube Extender Must be purchased separately We recommend labeling the tubes and the QlAamp UCP Mini columns for use on the QlAvac 24 Plus vacuum system according to the scheme in Figure 3 in order to avoid the mix up of samples This figure can be photocopied and labeled with the names of the samples 18 QlAamp UCP PurePathogen Blood Handbook 04 2011 Run ID Figure 3 Labeling scheme for tubes and QlAamp UCP Mini columns for use on the QlAvac 24 Plus vacuum system QlAamp UCP PurePathogen Blood Handbook 04 2011 19 Protocol Purification of Pathogen Nucleic Acids Including Free circulating and Viral Nucleic Acids from 3 ml to 8 ml Fresh EDTA Treated Blood This protocol is for purification of bacterial fungal viral and free circulating acids with parallel depletion of human DNA from up to 8 ml of fresh EDTA treated human blood Things to do before starting E Equilibrate samples to room temperature 15 25 C M Set up the QlAvac 24 Plus as described on pages 17 20 E Heat a water bath or heating block to 60 C for use with 50 ml centrifuge in step 15 M Heat a heating block to 56 C for use with 2 ml collection tubes in step 23
11. 4 Plus and the optional QlAvac Connecting System are checked for tightness on a daily basis to ensure sufficient vacuum strength QlAamp UCP PurePathogen Blood Handbook 04 2011 15 Regulator gauge Figure 1 Schematic diagram of the Vacuum Regulator Table 1 Chemical resistance properties of QlAvac 24 Plus Resistant to Acetic acid Chaotropic salts Chlorine bleach Chromic acid Concentrated alcohols Hydrochloric acid SDS Sodium chloride Sodium hydroxide Tween 20 Urea Not resistant to Benzene Chloroform Ethers Phenol Toluene 16 QlAamp UCP PurePathogen Blood Handbook 04 201 1 Setup of the QlAvac Plus vacuum manifold 1 Connect the QlAvac 24 Plus to a vacuum source If using the QlAvac Connecting System connect the system to the manifold and vacuum source as described in Appendix A of the QlAvac 24 Plus Handbook Note Close the outlets of the QlAvac 24 Plus using the plugs which are provided with the system or by closing the VacValves Apply vacuum using vacuum pump and ensure that pressure of 800 to 900 mbar is achieved Repeat this procedure every day before operation of the QlAvac system Insert a VacValve optional into each luer slot of the QlAvac 24 Plus that is to be used see Figure 2 Close unused luer slots with luer plugs or close the inserted VacValve VacValves should be used if flow rates of samples differ significantly to ensure consistent vacuum Insert a VacConn
12. April 2011 QlAamp UCP PurePathogen Blood Handbook For simultaneous purification of bacterial fungal viral and free circulating nucleic acids from whole blood 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 5 Intended Use 5 Safety Information 6 Quality Control 7 Introduction 8 Principle and procedure 8 Description of protocols 12 Equipment and Reagents to Be Supplied by User 13 Important Notes 14 Preparation of buffers and reagents 14 QlAvac 24 Plus 14 Protocol E Purification of Pathogen Nucleic Acids Including Free circulating and Viral Nucleic Acids from 3 ml to 8 ml Fresh EDTA Treated Blood 20 E Purification of Pathogen DNA from 3 ml to 8 ml Freeze Thawed EDTA Blood 24 Troubleshooting Guide 27 References 31 Appendix A General Remarks 31 Ordering Information 32 SESE QlAamp UCP PurePathogen Blood H
13. Blood Handbook 04 201 1 Note Steps 6a 6d are optional steps that can be performed to further decrease the content of background human DNA 6a Add 500 ul RNase free water to the centrifuge tube resuspend the microbial pellet by pipetting and transfer the whole volume into a fresh 1 5 ml centrifuge tube 6b Centrifuge the 1 5 ml tube for 5 min at 14 000 g in a microcentrifuge and discard the whole supernatant 6c Add 500 pl RNase free water and resuspend the microbial pellet by pipetting 6d Add 250 ul buffer and proceed with step 8 7 Add 250 ul of Buffer APR to the centrifuge tube and resuspend the microbial pellet by pipetting 8 Transfer the contents of the cell fraction buffer mixture 500 800 pl into a fresh Pathogen Lysis Tube L and close the tube tightly 9 Vortex the Pathogen Lysis Tube L according to steps 9a or 9b 9a Place the Pathogen Lysis Tube L on the Microtube foam insert and vortex for 10 min at maximum speed 9b Place the Pathogen Lysis Tube L on a TissueLyser LT and disrupt for 10 min at 50 Hz or use the FastPrep 24 instrument and apply a velocity of 6 5 m s for 2 x 45 s with a 5 min intermission 10 Remove the Pathogen Lysis Tube L from the vortexer and incubate the tube for 3 min at room temperature 15 25 C to reduce the foam in the tube Do not centrifuge 11 Carefully transfer the supernatant from the Pathogen Lysis Tube L into a fresh 15 ml tube while carefully keeping the glass beads fr
14. IAGEN spin columns in parallel Samples and wash solutions are drawn through the column membranes by vacuum instead of centrifugation providing greater speed and reduced hands on time in purification procedures In combination with the QlAvac Connecting System optional the QlAvac 24 Plus can be used as a flow through system The sample flow through is collected in a separate waste bottle For maintenance of the QlAvac 24 Plus refer to the handling guidelines in the QlAvac 24 Plus Handbook Contains chaotropic salt See page 6 for safety information t Contains sodium azide as a preservative 14 QlAamp UCP PurePathogen Blood Handbook 04 2011 Processing QlAamp UCP Mini columns on the QlAvac 24 Plus QlAamp UCP Mini columns are processed on the QlAvac 24 Plus using disposable VacConnectors and reusable VacValves VacValves optional are inserted directly into the luer slots of the QlAvac 24 Plus manifold and ensure a steady flow rate facilitating parallel processing of samples of different types e g blood and body fluids volumes or viscosities They should be used if sample flow rates differ significantly in order to ensure consistent vacuum VacConnectors are disposable connectors that fit between QlAamp UCP Mini columns and VacValves or between the QlAamp UCP Mini columns and the luer slots of the QlAvac 24 Plus They prevent direct contact between the spin column and VacValve during purification thereby avoiding any cross cont
15. Kit is designed to reduce this inhibition by depletion of human genomic DNA The QlAamp UCP PurePathogen Blood Kit procedure comprises the following steps 1 Separation of blood plasma from cellular fraction 2 Lysis of blood cells and depletion of human DNA within cellular fraction 3 Mechanical pre lysis of bacteria and fungi 4 Combination of pre lysed pathogens with plasma 5 Purification of pathogen nucleic acid lyse bind wash elute In the first step the plasma layer containing viruses and free circulating nucleic acids is separated from the cellular fraction through centrifugation and then kept for later use Next after specific lysis of the blood cells the microbial cells are enriched by centrifugation followed by a mechanical pre lysis step that enables the efficient disruption of difficult to lyse bacteria and fungi cells The resulting cell lysate is then combined with the plasma fraction containing the viral and free circulating nucleic acids To co purify viral and free circulating nucleic acids blood samples must be fresh However purification of bacterial and fungal nucleic acids from up to 8 ml of thawed blood samples is possible when using the dedicated protocol for freeze thawed blood samples see Protocol Purification of Pathogen DNA from 3 ml to 8 ml Freeze Thawed EDTA Blood page 24 8 QlAamp UCP PurePathogen Blood Handbook 04 2011 The QlAamp UCP PurePathogen Blood Protocols use extension tubes a
16. a Orders 86 21 3865 3865 Fax 86 21 3865 3965 Technical 800 988 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 UK Orders 01293 422 911 Fax 01293 422 922 Technical 01293 422 999 CLIII USA
17. amination between samples VacConnectors are discarded after a single USE Handling guidelines for the QlAvac 24 Plus Always place the QlAvac 24 Plus on a secure bench top or work area If dropped the QlAvac 24 Plus manifold may crack Always store the QlAvac 24 Plus clean and dry For cleaning procedures see the QlAvac 24 Plus Handbook The components of the QlAvac 24 Plus are not resistant to certain solvents Table 1 If these solvents are spilled on the unit rinse it thoroughly with water To ensure consistent performance do not apply silicone or vacuum greas to any part of the QlAvac 24 Plus manifold e Always use caution and wear safety glasses when working near a vacuum manifold under pressure Contact QIAGEN Technical Services or your local distributor for information concerning spare or replacement parts The vacuum pressure is the pressure differential between the inside of the vacuum manifold and the atmosphere standard atmospheric pressure 1013 millibar or 760 mm Hg and can be measured using the QlAvac Connecting System or a vacuum regulator see Figure 1 The protocols require a vacuum pump capable of producing a vacuum or 800 to 900 mbar e g QIAGEN Vacuum Pump Higher vacuum pressures must be avoided Use of vacuum pressures lower than recommended may reduce nucleic acid yield and purity and increase the risk of clogged membranes Make sure that the connections between the vacuum pump QlAvac 2
18. andbook 04 2011 3 Kit Contents QlAamp UCP PurePathogen Blood Kit 10 Catalog no 50112 Number of preps 10 QlAamp UCP Mini Columns 10 Collection Tubes 2 0 ml 50 Pathogen Lysis Tubes L 10 Tube Extenders 20 ml 10 Elution Tubes 1 5 ml 50 VacConnectors 50 Buffer APR 14 ml Buffer APL1t 56 ml Buffer APL2 46 ml Buffer APB1 concentrate 3 x 20 ml Buffer APWT concentrate 6 ml Buffer APW2 concentrate 4 ml Buffer AVE 3 vials Reagent DX vial RNase free water 15 ml Proteinase K 6 ml Handbook 1 Contains sodium azide as a preservative t Contains small amounts of guanidine thiocyanate Contains chaotropic salt See page 6 for safety information 4 QlAamp UCP PurePathogen Blood Handbook 04 201 1 Storage QlAamp UCP Mini columns should be stored at 2 8 C upon arrival However short term storage up to 4 weeks at room temperature 15 25 C does not affect their performance All buffers can be stored at room temperature 15 25 C The QlAamp UCP PurePathogen Blood Kit contains a ready to use Proteinase K solution which is dissolved in a specially formulated storage buffer The Proteinase K is stable for up to 1 year after delivery when stored at room temperature 15 25 C To prolong the lifetime of Proteinase K storage at 2 8 C is recommended The QlAamp UCP Mini Columns and all buffers within the QlAamp UCP PurePathogen Blood Kit undergo proprietary DNA decontamination processes and are cert
19. ector into each VacValve see Figure 2 Perform this step directly before starting the purification to avoid exposure of VacConnectors to potential contaminants in the air Place the QlAamp UCP Mini columns into the VacConnectors on the manifold see Figure 2 Note Save the collection tube from the blister pack for use in the purification protocol Insert a tube extender 20 ml into each QlAamp UCP Mini column see Figure 2 Note Make sure that the tube extender is firmly inserted into the QlAamp UCP Mini column in order to avoid leakage of sample For nucleic acid purification follow the instructions in the protocols Discard the VacConnectors appropriately after use Leave the lid of the QlAamp UCP Mini column open while applying vacuum Switch off the vacuum between steps to ensure that a consistent even vacuum is applied during processing For faster vacuum release a vacuum regulator should be used see Figure 1 Note Each VacValve can be closed individually when the sample is completely drawn through the spin column allowing parallel processing of samples of different volumes or viscosities After processing samples clean the QlAvac 24 Plus see Cleaning and Decontaminating the QlAvac 24 Plus in the QlAvac 24 Plus Handbook Note Buffers APL APL2 APB1 and APW1 are not compatible with disinfecting agents containing bleach See page 6 for safety information QlAamp UCP PurePathogen Blood Handbook 04 20
20. embrane Close the lid and incubate at room temperature for 3 min IMPORTANT Ensure that the elution buffer is equilibrated to room temperature Elution volume is flexible and can be adapted according to the requirements of downstream applications The recovered eluate volume will be up to 5 ul less than the elution volume applied onto the column If elution is done in small volumes 100 ul the elution buffer has to be dispensed onto the center of the membrane for complete elution of bound QlAamp UCP PurePathogen Blood Handbook 04 201 1 DNA Small elution volumes may lead to inhibition of downstream reactions Determine the optional ratio of elution volume and the volume of eluate suitable for your downstream reaction and reduce or increase the elution volume accordingly 25 Centrifuge at full speed 20 000 x g 14 000 rpm for 1 min to elute the DNA ee QlAamp UCP PurePathogen Blood Handbook 04 2011 23 Protocol Purification of Pathogen DNA from 3 ml to 8 ml Freeze Thawed EDTA Blood This protocol is for purification of bacterial and fungal nucleic acids with parallel depletion of human DNA from up to 8 ml thawed EDTA treated human blood Things to do before starting M Equilibrate samples to room temperature 15 25 C M Set up the QlAvac 24 Plus as described on pages 17 20 E Heat a water bath or heating block to 60 C for use with 50 ml centrifuge in step 15 E Heat a heating block to 56 C for use with 2 ml col
21. er of the membrane for complete elution of bound DNA Small elution volumes moy lead to inhibition of downstream reactions Determine the optional ratio of elution volume and the volume of eluate suitable for your downstream reaction and reduce or increase the elution volume accordingly Centrifuge at full speed 20 000 x g 14 000 rpm for 1 min to elute the DNA QlAamp UCP PurePathogen Blood Handbook 04 201 1 Troubleshooting Guide This troubleshooting guide may be helpful in solving any problems that may arise For more information see also the Frequently Asked Questions Page at our Technical Support Center www qgiagen 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 giagen com Comments and suggestions Little or no pathogen DNA in the eluate a Primary blood tube Anticoagulants other than EDTA may lead to contains an accelerated DNA degradation compared to anticoagulant other EDTA blood Repeat the purification procedure than EDTA with new samples b Pathogen cells may The pathogen cells may have been discarded have been discarded along with the supernatant containing the along with the human DNA Be careful not to discard the supernatant microbial cells c Inefficient mechanical Make sure that the Pathogen L
22. escription of protocols Different protocols are provided for human blood fresh or thawed blood The Protocol Purification of Pathogen Nucleic Acids Including Free circulating and Viral Nucleic Acids from 3 ml to 8 ml Fresh EDTA Treated Blood page 20 is for processing 3 8 ml fresh blood The Protocol Purification of Pathogen DNA from 3 ml to 8 ml Freeze Thawed EDTA Blood page 24 is designed for purification of 3 8 ml thawed human blood 12 QlAamp UCP PurePathogen Blood Handbook 04 201 1 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 HE Pipets adjustable M Sterile pipet tips pipet tips with aerosol barriers are recommended to help prevent cross contamination HE Water bath or heating block capable of holding 50 ml centrifuge tubes at 60 C E Heating block or similar at 56 C capable of holding 2 ml collection tubes M Microcentrifuge M Microcentrifuge tubes 2 ml M Vortexer with Microtube foam insert cat no 504 0234 00 or TurboMix Attachment cat no SI 0563 both available from Scientific Industries TissueLyser LT cat no 85600 which is available from QIAGEN or FASTPREP 24 cat no 116003500 which is available from MP Biomedicals Wi Sterile 15 ml and 50 ml centrifuge tubes
23. g a Vacuum pressure of 800 900 mbar not reached QlAamp UCP PurePathogen Blood Handbook 04 2011 Comments and suggestions Ensure that Buffers APW1 and APW2 are used in the correct order in the protocol Repeat the purification procedure with a new sample Adjust the volume of eluate added to the downstream reaction Use recommended drying step in the relevant protocol The vacuum manifold is not tightly closed Close the outlets of the QlAvac 24 Plus using the plugs which are provided with the system or by closing the VacValves Apply vacuum using vacuum pump and ensure that pressure of 800 to 900 mbar is achieved Repeat this procedure every day before operation of the QlAvac system Gasket of QlAvac lid has worn out Check the seal of the manifold visually and replace it if necessary VacValves have worn out Remove all VacValves and insert VacConnectors directly into the luer extensions Insert QlAamp UCP Mini columns into VacConnectors close the lid of the columns and switch on vacuum Check if vacuum pressure is reached Replace VacValves if necessary Connection to vacuum pump is leaky Close all luer extensions with luer caps and switch on the vacuum pump Check if vacuum pressure is stable after the pump is switched on and the vacuum regulator valve is closed Exchange the connections between pump and vacuum manifold if necessary If the vacuum pressure is still not reached replace the vacuum pump with a
24. ified to have no detectable contaminating microbial DNA at the time of delivery Intended Use The QlAamp UCP PurePathogen Blood Kit is intended for molecular biology applications This product is not intended for the diagnosis prevention or treatment of a disease All due care and attention should be exercised in the handling of the products We recommend all users of QIAGEN products to adhere to the NIH guidelines that have been developed for recombinant DNA experiments or to other applicable guidelines mmu E M M M M ie QlAamp UCP PurePathogen Blood Handbook 04 201 1 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 or acidic solutions directly to waste containing Buffer APL1 Buffer APL2 Buffer APB1 or Buffer APW1 Buffer APL1 Buffer APL2 Buffer APB1 and Buffer APW1 contain guanidine salts which can form highly reactive compounds when combined with bleach If liquid containing these buffers is spilt clean with suitable laboratory detergent and water
25. lection tubes in step 23 E Ensure that Buffer APR Buffer APB1 Buffer APW1 and Buffer APW2 have been prepared according to the instructions on page 14 Procedure 1 Place 3 to 8 ml of frozen then thawed blood into a 15 ml centrifuge tube not provided Close the cap and centrifuge 5 000 x g for 10 min 2 Open the cap and carefully pipette and discard the liquid supernatant leaving approximately 500 ul cell fraction in the bottom of the tube 3 Add 4 ml Buffer APL1 to the remaining cell fraction from step 2 and mix vigorously by pulse vortexing for 30 s Buffer APL1 specifically lyses human blood cells while microbial cells stay intact In some cases precipitates might occur which do not dissolve even after proper vortexing Proceed with the procedure as described 4 Incubate the cell fraction buffer mixture for 10 min at room temperature 15 25 C 5 Close the tube and centrifuge cell fraction buffer mixture for 10 min 5 000 x g 6 Carefully discard the supernatant but save the microbial cell pellet Note Do not vigorously shake the tube while discarding the supernatant A small fraction of liquid 300 500 ul flowback will not disturb the process Too vigorous shaking or decanting may lead to the loss of the microbial cells Due to low pathogen titers the microbial pellet is not visible in most cases Proceed with the process even if you do not observe the pellet at the bottom of the tube 24 QlAamp UCP PurePathogen
26. luded within this Kit except as described in the QlAamp UCP PurePathogen Blood 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 C exp 09e NS 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 S For updated license terms see www giagen com 2011 QIAGEN all rights reserved www qiagen 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 Chin
27. n Blood Handbook 04 2011 11 For downstream applications that require a larger starting volume or for multiple analysis the elution volume can be increased up to 200 ul However an increase in elution volume will decrease the concentration of nucleic acids in the eluate The eluate volume recovered can be up to 5 ul less than the volume of elution buffer applied to this column for example an elution volume of 20 ul results in lt 15 ul final eluate The volume of eluate recovered depends on the nature of the sample Eluted DNA is collected in 1 5 ml microcentrifuge tubes provided If the purified nucleic acids are to be stored for up to 24 hours storage at 2 8 C is recommended For periods of storage longer than 24 hours storage at 15 to 30 C is recommended Yield and size of nucleic acids Although the QlAamp UCP PurePathogen Blood Kit significantly reduces the amount of human nucleic acids to prevent downstream inhibition the eluates still contain human DNA Yields of pathogen nucleic acids isolated from biological samples are normally below 1 ug and therefore cannot be distinguished by a spectrophotometer The size distribution of pathogen nucleic acids purified using this procedure can be checked by agarose gel electrophoresis and hybridization to a target specific labeled probe Sambrook J and Russell D W 2001 Molecular Cloning a Laboratory Manual 3 ed Cold Spring Harbor NY Cold Spring Harbor Laboratory Press D
28. nd vacuum processing on the QlAvac 24 Plus to enable starting sample volumes of up to 8 ml blood and flexible elution volumes from 50 ul to 200 ul The robust procedure helps to eliminate sample to sample cross contamination and increases user safety when handling potentially infectious samples The simple process is highly suited for simultaneous processing of multiple samples and provides purified nucleic acids in less than 2 hours per 10 samples QlAamp UCP PurePathogen Blood Handbook 04 2011 9 10 QlAamp UCP PurePathogen Procedure 1 Volume blood lysis buffer 7 Discard supernatant 7 Mechanical pre lysis of microbial Pellet Pure nucleic acids QlAamp UCP PurePathogen Blood Handbook 04 2011 Sample volumes and material The QlAamp UCP PurePathogen Blood Kit has been optimized for large sample volumes of up to 8 ml blood and comes with specific protocols for fresh as well as thawed blood Lysing samples Due to their rigid cell wall bacterial and fungal cells require a mechanical pre lysis step to ensure complete lysis Samples are suspended with Buffer APR transferred to Pathogen Lysis Tubes and vortexed Samples are then lysed under highly denaturing conditions at elevated temperatures in the presence of Proteinase K and Buffer APL2 to ensure lysis of viruses inactivation of nucleases and complete release of nucleic acids from pre lysed microbial cells Adsorption to the QlAamp Mini membrane Binding co
29. nditions are adjusted by adding Buffer APB1 to allow optimal binding of the pathogen nucleic acids to the membrane Lysates are then transferred onto a QlAamp UCP Mini column and the pathogen nucleic acids are adsorbed onto the silica membrane as the lysate is drawn through by vacuum pressure Salt and pH conditions ensure that proteins and other contaminants which can inhibit downstream reactions are not retained on the QlAamp Mini membrane A vacuum manifold e g the QlAvac 24 Plus with the QlAvac Connecting System and a vacuum pump capable of producing a vacuum of 800 to 900 mbar e g QIAGEN Vacuum Pump are required for the protocol A vacuum regulator should be used for easy monitoring of vacuum pressures and convenient vacuum releases Removal of residual contaminants Nucleic acids remain bound to the membrane while contaminants are efficiently washed away during 3 wash steps In a single step highly pure pathogen nucleic acids are eluted in Buffer AVE equilibrated to room temperature Elution of purified nucleic acids from pathogens Elution is performed using Buffer AVE The elution volume can be as low as 50 ul If higher nucleic acid concentrations are required the elution volume can be reduced to as low as 20 ul Low elution volume leads to highly concentrated eluates For downstream applications that require small starting volumes a more concentrated eluate may increase assay sensitivity QlAamp UCP PurePathoge
30. om being transferred into the tube 12 Wash the glass beads by adding 500 ul RNase free water to the Pathogen Lysis Tube L Close the lid Vortex open lid and transfer 500 ul of the supernatant to the tube from step 11 Discard the Pathogen Lysis Tube 13 Add 100 pl Proteinase K to the 15 ml tube close the cap and mix by pulse vortexing for 10 s 14 Add 800 pl of Buffer APL2 close the cap and mix by pulse vortexing for 10s 15 Incubate tube at 60 C for 15 min 16 Briefly spin the tube to remove drops from the inside of the lid 17 Add 3 2 ml of Buffer APB1 to the lysate close the cap and mix thoroughly by pulse vortexing for 15 30 s QlAamp UCP PurePathogen Blood Handbook 04 2011 25 18 19 20 21 22 23 24 25 26 Carefully apply the lysate from step 17 into the extension tube of the QlAamp UCP Mini column Switch on the vacuum pump When all lysates have been drawn through the columns completely switch off the vacuum pump and release the pressure to 0 mbar Apply 600 ul of Buffer APW1 to the QlAamp UCP Mini column Carefully remove and discard the extension tube and switch on the vacuum pump After all of Buffer APW1 has been drawn through the Mini column switch off the vacuum pump and release the pressure to O mbar Note To avoid cross contamination be careful not to move the tube extenders over neighboring QlAamp UCP Mini Columns Apply 750 ul of Buffer APW2 to the QlAamp UCP Mini
31. on of liquid 300 500 ul flowback will not disturb the process Too vigorous shaking or decanting may lead to the loss of the microbial cells Due to low pathogen titers the microbial pellet is not visible in most cases Proceed with the process even if you do not observe the pellet at the bottom of the tube Note Steps 6a 6d are optional steps that can be performed to further decrease the content of background human DNA 6a Add 500 ul RNase free water to the centrifuge tube resuspend the microbial pellet by pipetting and transfer the whole volume into a fresh 1 5 ml centrifuge tube 6b Centrifuge the 1 5 ml tube for 5 min at 14 000 g in a microcentrifuge and discard the whole supernatant 6c Add 500 pl RNase free water and resuspend the microbial pellet by pipetting 6d Add 250 ul buffer and proceed with step 8 7 Add 250 ul of Buffer APR to the centrifuge tube and resuspend the microbial pellet by pipetting 8 Transfer the contents of the cell fraction buffer mixture 500 800 yl into a fresh Pathogen Lysis Tube L and close the tube tightly 9 Vortex the Pathogen Lysis Tube L according to steps 9a or 9b 9a Place the Pathogen Lysis Tube L on the Microtube foam insert and vortex for 10 min at maximum speed 9b Place the Pathogen Lysis Tube L on a TissueLyser LT and disrupt for 10 min at 50 Hz or use the FastPrep 24 instrument and apply a velocity of 6 5 m s for 2 x 45s with a 5 min intermission 10 Remove the Pathogen L
32. stronger one If using the optional QlAvac Connecting System check if assembly is correct according to instructions 29 Comments and suggestions b Inefficient proteinase If Proteinase K was subjected to elevated digestion temperature for a prolonged time it can lose activity Repeat the procedure using new samples and fresh Proteinase K c Clogged QlAamp UCP Close the VacValve if used and carefully Mini column remove the whole assembly consisting of tube extender QlAamp UCP Mini column VacConnector and VacValve from the QlAvac 24 Plus manifold Carefully transfer the remaining sample lysate from the tube extender to a new 50 ml tube Remove the QlAamp UCP Mini column from the assembly see above place it in a 2 ml collection tube and spin it at full speed for 1 minute or until sample has completely passed through the membrane Re assemble the QlAamp UCP Mini column with Tube Extender VacConnector and optional VacValve Transfer the remaining sample lysate into the Tube Extender switch on the vacuum pump open the VacValve and pass the remaining lysate through the QlAamp UCP Mini column Repeat the above procedure if the QlAamp UCP Mini Column continues to clog 30 QlAamp UCP PurePathogen Blood Handbook 04 201 1 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
33. with the QlAvac 24 19408 Plus VacConnectors 500 500 disposable connectors for use with 19407 QlAamp spin columns on luer slots or VacValves Vacuum Pump Universal vacuum pump capacity 34 84010 liters min 8 mbar vacuum abs 84000 84020 QlAvac Connecting System to connect vacuum manifold 19419 System with vacuum pump includes Tray Waste Bottles Tubing Couplings Valve Gauge 24 VacValve Collection Tubes 2 ml 1000 Collection Tubes 2 ml 19201 QIAGEN Proteinase K 2 ml gt 600 mAU mnl solution 19131 2 ml QIAGEN Proteinase K 10 ml 2600 mAU ml solution 19201 10 ml US and Canada t Japan Rest of world n Su aa 32 QlAamp UCP PurePathogen Blood Handbook 04 2011 Notes QlAamp UCP PurePathogen Blood Handbook 04 2011 33 Notes 34 QlAamp UCP PurePathogen Blood Handbook 04 2011 Trademarks QIAGEN QlAamp QIAGEN Group FASTPREP MP Biomedicals LLC Vortex Genie Scientific Industries Inc TurboMix Bete Fog Nozzle Inc Limited License Agreement Use of this product signifies the agreement of any purchaser or user of the QlAamp UCP PurePathogen Blood Kit to the following terms 1 The QlAamp UCP PurePathogen Blood Kit may be used solely in accordance with the QlAamp UCP PurePathogen Blood 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 inc
34. ysis Tube L from the vortexer and incubate the tube for 3 min at room temperature 15 25 C to reduce the foam in the tube Do not centrifuge 11 Carefully transfer the supernatant from the Pathogen Lysis Tube L into the plasma fraction tube while carefully keeping the glass beads from being transferred into the plasma fraction from step 2 This step combines the mechanically pre lysed microbial cells with the free circulating nucleic acids and the viruses 12 Wash the glass beads by adding 500 ul RNase free water to the Pathogen Lysis Tube L Close the lid Vortex and transfer 500 pl of the supernatant to the plasma mixture from step 11 Discard the Pathogen Lysis Tube 13 Add 500 pl Proteinase K to the tube close the cap and mix by pulse vortexing for 10 s QlAamp UCP PurePathogen Blood Handbook 04 2011 21 14 15 16 17 18 19 20 21 22 23 24 22 Add 4 ml of Buffer APL2 to the tube close the cap and mix by pulse vortexing for 10 s Incubate tube at 60 C for 15 min Briefly spin the tube to remove drops from the inside of the lid Add 16 ml of Buffer APB1 to the lysate close the cap and mix thoroughly by pulse vortexing for 15 30 s Carefully apply the lysate from step 17 into the extension tube of the QlAamp UCP Mini column Switch on the vacuum pump When all lysates have been drawn through the columns completely switch off the vacuum pump and release the pressure to 0 mbar
35. ysis Tube has been lysis of pathogens vortexed for 10 min at maximal speed using a Microtube foam insert of a Vortex Genie a TissueLyser LT at 50 Hz or a FASTPREP 24 instrument with 2 x 45 s with a 5 min intermission d Low percentage Repeat the purification procedure with new ethanol used instead of samples and 96 100 ethanol Do not use 96 100 denatured alcohol which contains other substances such as methanol or methylethylketone e Buffer APB prepared Check that Buffer APB1 concentrate was incorrectly reconstituted with the correct volume of isopropanol not ethanol see page 14 f Buffer APW1 or Buffer Check that Buffer APW1 and Buffer APW2 APW2 prepared concentrates were diluted with the correct volume incorrectly of ethanol see page 14 Repeat the purification procedure with new samples QlAamp UCP PurePathogen Blood Handbook 04 2011 27 Comments and suggestions g Buffer APW1 or Buffer Check that Buffer APW1 and Buffer APW2 APW2 prepared with concentrates were diluted with 96 100 ethanol 70 ethanol see page 14 Repeat the purification procedure with new samples h QlAamp UCP Mini After addition of Buffer AVE the QlAamp UCP column not incubated Mini column should be incubated at room at room temperature temperature for 3 min 15 25 C for 3 min High human DNA background a Samples were kept for Blood cells may disintegrate and release an extended time genomic DNA into the plasma diluting the
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