Sample & Assay Technologies QIAamp® cador® Pathogen
Contents
1. Mechanical Disruption of Tissue 26 E Pretreatment T2 Enzymatic Digestion of Tissue 27 E Pretreatment T3 Rapid Partial Disruption of Tissue 29 Pretreatment T4 Organic Extraction for Difficult Tissue 30 Troubleshooting Guide 32 References 35 Ordering Information 36 QlAamp cador Pathogen Mini Handbook 06 2012 3 Kit Contents QlAamp cador Pathogen Mini Kit 50 250 Catalog no 54104 54106 Number of preps 50 250 QlAamp Mini Columns 50 250 Collection Tubes 2 ml 200 1000 Buffer VXL 6 ml 30 ml Buffer ACB t concentrate 12 ml 60 ml QIAGEN Proteinase K 1 25 ml 6 ml Carrier RNA poly A 310 ug 310 ug Buffer AW 1 concentrate 19 ml 98 ml Buffer AW2 concentrate 17 ml 81 ml Buffer AVES 20 ml 2 x 20 ml Quick Start Protocol 1 1 Contains a chaotropic salt Take appropriate laboratory safety measures and wear gloves when handling Not compatible with disinfectants containing bleach See page 6 for safety information t Before using for the first time add isopropanol as indicated on the bottle to obtain a working solution Before using for the first time add ethanol 96 100 as indicated on the bottle to obtain a working solution Contains sodium azide as a preservative 4 QlAamp cador Pathogen Mini Handbook 06 2012 Storage QlAamp Mini columns and buffers can be stored dry at room temperature 15 25 C until the expiration date on the kit box without affecting performance Lyophilized ca2. However depending on the starting material and the target pathogen one of the pretreatment protocols may be needed Table 1 on page 8 provides an overview of which pretreatment protocols are suited to which starting material and pathogen combinations M Nucleic acid purification protocol page 20 Mi Pretreatment protocols pages 23 31 PP naaa aa aa a RP QlAamp cador Pathogen Mini Handbook 06 2012 7 Table 1 Pretreatment protocols for fluid and tissue samples Sample Target Pretreatment Page Fluids Viral RNA and DNA 20 e g whole blood es of COSES serum plasma acera swab or wash fluid pretreated tissue Whole blood or DNA of difficult to lyse Pretreatment B1 23 pretreated tissue bacteria for difficult to lyse bacteria in whole blood or pretreated tissue Serum plasma DNA of difficult to lyse Pretreatment B2 24 swabs washes bacteria for difficult to lyse bacteria in body cavity fluids cell free fluidst urine High volume DNA of easy to lyse Pretreatment B3 25 cell free fluids for bacteria for easy to lyse bacteria in increased high volume cell free fluids sensitivity Tissue Viral RNA and DNA Pretreatment T1 26 Sde liver Saleen mechanical disruption of tissue kidney Viral DNAS Pretreatment T2 27 lymph node bacterial DNA enzymatic digestion of tissue Viral RNA and DNA Pretreatment T3 29 bacterial DNA rapid partial disruption of tissue Difficult tissue Viral RNA and DNA Pretreatment T4 30 EG brani
3. The choice of pretreatment depends on the workflow focus and is to be followed by nucleic acid purification Table 1 on page 8 summarizes the pretreatments and their applications Some of the pretreatments may require additional components see page 12 Pretreatment for extraction of DNA of difficult to lyse bacteria Treatment with chemicals and proteinase K is sufficient for complete lysis in the case of many Gram negative bacteria but the cell walls of Gram positive bacteria and some Gram negative bacteria must be disrupted by additional methods For maximal lysis efficiency when working with difficult to lyse bacteria we recommend mechanical disruption using Pathogen Lysis Tubes which contain glass beads The tubes must be ordered separately for ordering information see page 36 For pretreatment of difficult to lyse bacteria in whole blood use Pretreatment B1 page 23 For pretreatment of difficult to lyse bacteria from cell free fluid samples such as serum plasma swabs washes body cavity fluids and urine use Pretreatment B2 page 24 The processing of samples with high inhibitor contents such as urine may require a reduction in sample input volume QlAamp cador Pathogen Mini Handbook 06 2012 9 For pretreatment of difficult to lyse bacteria in tissue samples first use one of the tissue pretreatments see pages 9 and 23 31 followed by Pretreatment B1 page 23 Optional pretreatment for easy to lyse bacteria
4. on of ae organic extraction for difficult pancreas acteria tissue adipose tissue Gram positive bacteria are difficult to lyse due to their rigid cell wall Many Gram negative bacteria are easy to lyse but some are difficult to lyse and will also benefit from Pretreatment B1 or B2 t Not suitable for whole blood Not suitable for bacterial DNA due to centrifugation step see page 26 Not suitable for viral RNA as the lysis conditions do not sufficiently conserve RNA integrity 1 For difficult to lyse bacteria subsequently use Pretreatment B1 page 23 Not suitable for difficult to lyse bacteria as they would be lost during the centrifugation step 8 QlAamp cador Pathogen Mini Handbook 06 2012 Nucleic acid purification protocol The protocol Purification of Pathogen Nucleic acids from Fluid Samples page 20 is optimized for purification of viral RNA and DNA and the DNA of easy to lyse bacteria from up to 200 ul of fluid material Suitable starting materials for direct processing using this method include BE Whole blood E Serum E Plasma E Body cavity fluids e g peritoneal synovial cerebrospinal Mi Liquid extracts from swabs e g nasal pharyngeal and cloacal swabs H Wash fluids e g from bronchoalveolar lavages Mi Other fluids such as urine Pretreatments The various pretreatments included in this handbook are optimized for specific combinations of starting material and target pathogen
5. 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 2011 2012 QIAGEN all rights reserved www qiagen com Australia techservice au giagen com Austria techservice at qiagen com Belgium techservice bnI qiagen com Brazil suportetecnico brasil giagen com Canada techservice ca giagen com China techservice cn qiagen com Denmark techservice nordic qiagen com Finland techservice nordic giagen com France techservice fr qiagen com Germany techservice de giagen com Hong Kong techservice hk qiagen com India techservice india giagen com Ireland techservice uk qiagen com Italy techservice it giagen com Japan techservice pOqiagen com Korea South techservice kr giagen com Luxembourg techservice bnlOqiagen com Mexico techservice mx qiagen com The Netherlands techservice bnl giagen com Norway techservice nordic giagen com Singapore techservice sg qiagen com Sweden techservice nordic qiagen com Switzerland techservice ch giagen com amp O UK techservice uk giagen com USA techservice us qiagen com QIAGEN aaa Sample 8 Assay Technologies
6. Microtube Foam Insert and vortex for 10 min at maximum speed Alternatively the Pathogen Lysis Tube may be processed on a TissueLyser LT for 10 min at 50 Hz or on a FastPrep 24 by applying a velocity of 6 5 m s for two 45 s periods with a 5 min resting time between them 4 Remove the Pathogen Lysis Tube from the vortexer and briefly centrifuge the tube to remove drops from the inside of the lid Use 200 pl of the supernatant as starting material for the protocol Purification of Pathogen Nucleic acids from Fluid Samples page 20 aaa a QlAamp cador Pathogen Mini Handbook 06 2012 23 Pretreatment B2 for Difficult to Lyse Bacteria in Cell Free Fluids This pretreatment is for the extraction of bacterial DNA of difficult to lyse bacteria from cell free fluids such as serum Important points before starting E Buffer ATL and Pathogen Lysis Tubes L or S with glass beads including Reagent DX must be ordered separately for ordering information see page 36 Note The choice of L or S tubes depends on the bacterial target E Buffer ATL may form precipitates upon storage If necessary warm to 56 C until the precipitates have fully dissolved Things to do before starting E Before use add 100 ul Reagent DX to 15 ml Buffer ATL If smaller amounts are needed transfer 1 5 ml of Buffer ATL into a sterile 2 ml vial and add 10 ul Reagent DX Mix well after addition of Reagent DX After preparation the mixture is stable for 6
7. Notes Starting material Do not overload the QlAamp Mini column as this can lead to impaired nucleic acid extraction and or performance in downstream assays For samples with very high host nucleic acid contents e g for certain tissues such as spleen or blood samples with highly increased cell counts use less than the maximum amount of sample recommended in the protocol or pretreatments In some downstream applications such as PCR and RT PCR very high background concentrations of nucleic acids may impair the reaction Use appropriate controls e g an internal control to verify successful PCR amplification Avoid transferring material to the QlAamp Mini column that could subsequently clog the membrane e g blood clots solid tissue swab fibers Highly viscous fluids may require a treatment to reduce their viscosity to allow for efficient extraction of pathogen nucleic acids Please contact QIAGEN Technical Service for recommendations Avoid repeated thawing and freezing of samples since this may reduce nucleic acid yield and quality Animal whole blood Blood samples treated with EDTA citrate or heparin as anticoagulant can be used for nucleic acid purification Samples can be either fresh or frozen provided that they have not been frozen and thawed more than once After collection whole blood samples can be stored at 2 8 C for up to 6 hours For longer storage we recommend freezing aliquots at 20 C or 80 C We rec
8. When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information please consult the appropriate safety data sheets SDSs These are available online in convenient and compact PDF format at www giagen com safety where you can find view and print the SDS for each QIAGEN kit and kit component CAUTION DO NOT add bleach or acidic solutions directly to the sample preparation waste Buffer VXL and Buffer AW1 contain guanidine hydrochloride and Buffer ACB contains guanidine thiocyanate which can form highly reactive compounds if combined with bleach If liquid containing these buffers is spilled clean with suitable laboratory detergent and water If the spilled liquid contains potentially infectious agents clean the affected area first with laboratory detergent and water and then with 1 v v sodium hypochlorite 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 the QlAamp cador Pathogen Mini Kit is tested against predetermined specifications to ensure consistent product quality 6 QlAamp cador Pathogen Mini Handbook 06 2012 Introduction The QlAamp cador Pathogen Mini Kit enables the efficient purification of viral RNA
9. acids at 20 C or even 80 C in the case of RNA Handling RNA RNases are very stable and active enzymes that generally do not require cofactors to function Since RNases are difficult to inactivate and only minute amounts are sufficient to destroy RNA do not use any plasticware or glassware without first eliminating possible RNase contamination Great care should be taken to avoid inadvertently introducing RNases into the RNA sample during or after the purification procedure Preparing reagents Carrier RNA stock solution For use lyophilized carrier RNA should first be dissolved in Buffer AVE Add 310 ul Buffer AVE to the tube containing 310 ug lyophilized carrier RNA to obtain a stock solution of 1 ug ul Add this solution to Buffer VXL as described below Unused carrier RNA dissolved in Buffer AVE should be frozen in aliquots at 20 C Aliquots of carrier RNA should not be subjected to more than 3 freeze thaw cycles Adding carrier RNA to Buffer VXL We recommend adding carrier RNA to fluids containing a low amount of cells such as serum plasma swabs media and wash fluid Do not add carrier RNA to samples with a high cell content such as whole blood and tissue because high amounts of background nucleic acids may negatively influence downstream applications such as RT PCR Carrier RNA dissolved in Buffer AVE is added to Buffer VXL see protocols starting on page 20 so that 1 ug carrier RNA is present in each sample Note
10. and DNA and bacterial DNA from a broad range of animal samples including whole blood serum plasma swabs washes and tissue see Starting material on page 14 The extracted nucleic acids are free of proteins nucleases and other impurities and are ready for use in downstream applications such as real time PCR based pathogen identification The kit is not intended for host RNA or host DNA preparation Principle and procedure Samples are lysed under highly denaturing conditions at room temperature 15 25 C in the presence of proteinase K and Buffer VXL which together ensure the inactivation of nucleases Adding Buffer ACB adjusts the binding conditions for the copurification of DNA and RNA The lysate is then transferred to a QlAamp Mini column During centrifugation nucleic acids are adsorbed onto the silica membranes while contaminants pass through Two efficient wash steps remove the remaining contaminants and enzyme inhibitors and nucleic acids are eluted in Buffer AVE Performance is not guaranteed for every combination of starting material and pathogen species and must be validated by the user Some samples may require a pretreatment see Table 1 page 8 Description of protocols There are two types of protocol in this handbook Samples will either directly undergo nucleic acid purification or undergo pretreatment followed by nucleic acid purification Most sample types can be directly processed without pretreatment
11. filtrate If the lysate has not completely passed through the column after centrifugation centrifuge again at a higher speed up to 20 000 x g 14 000 rpm until the QlAamp Mini column is empty 9 Open the QlAamp Mini column and add 600 ul Buffer AW1 without wetting the rim Close the cap and centrifuge at 6000 x g 8000 rpm for 1 min Place the QlAamp Mini column in a clean 2 ml collection tube and discard the tube containing the filtrate 10 Open the QlAamp Mini column and add 600 ul Buffer AW2 without wetting the rim Close the cap and centrifuge at 6000 x g 8000 rpm for 1 min Place the QlAamp Mini column in a clean 2 ml collection tube and discard the tube containing the filtrate 11 Centrifuge at full speed 20 000 x g 14 000 rpm for 2 min to dry the membrane QlAamp cador Pathogen Mini Handbook 06 2012 21 12 Place the QlAamp Mini column in a clean 1 5 ml microcentrifuge tube not provided and discard the collection tube containing the filtrate Open the QlAamp Mini column and add 50 150 pl Buffer AVE to the center of the membrane Close the cap and incubate at room temperature 15 25 C for 1 min Centrifuge at full speed 20 000 x g 14 000 rpm for 1 min Important Ensure that the elution buffer is equilibrated to room temperature If elution is performed with a small volume lt 75 ul the elution buffer must be dispensed onto the center of the membrane for complete elution of bound RNA and DNA Elution volu
12. in high volume cell free fluids Optionally to increase the sensitivity of detection for DNA of easy to lyse bacteria from fluid samples containing no cells or a low amount of cells such as serum plasma other cell free body fluids swabs and washes the sample input volume can be increased Subsequently the bacteria can be concentrated by pelleting To use an increased input volume before the extraction of DNA of easy to lyse bacteria use Pretreatment B3 page 25 Pretreatment of tissue samples Mechanical or enzymatic disruption of tissue structure is a prerequisite to enable the extraction and purification of nucleic acids The protocols listed in this section are for tissue pretreatment in workflows focused on viral RNA and DNA and bacterial DNA The starting material is up to 25 mg of tissue such as liver lymph node spleen or kidney The choice of pretreatment depends on the various workflow demands the targeted pathogen type of tissue anticipated sensitivity of the application available laboratory equipment and acceptable time and effort The pretreatment protocols are suited for most types of tissue and pathogens However we recommend evaluating the suitability of these approaches for each new combination of tissue type and pathogen To extract viral RNA and viral DNA from tissue use Pretreatment T1 page 26 This pretreatment is not suitable for the extraction of bacterial DNA due to the centrifugation step which ma
13. lyse bacteria from tissues containing a high amount of lipids and or nucleases such as brain or pancreas Important point before starting Phenol and chloroform must be purchased separately We recommend using equilibrated phenol solution pH 8 e g Sigma cat no P4557 Ensure you are familiar with the safety requirements for these chemicals For more information please consult the appropriate safety data sheets SDSs available from the product supplier Use appropriate tubes with a safe lock lid Things to do before starting M Cool a microcentrifuge to 4 C for use in step 8 Procedure 1 Place up to 25 mg tissue in 2 ml microcentrifuge tubes each containing 1 stainless steel bead 5 mm mean diameter For tissues with a very high number of cells for a given mass of tissue e g spleen a reduced amount of starting material 5 10 mg should be used If working with fibrous tissues cutting the tissue into smaller pieces before starting disruption will improve disruption efficiency 2 Add 400 pl PBS or 0 9 NaCl solution and 200 ul phenol TE saturated pH 8 to each tube and close the lid tightly Place the tubes in the TissueLyser II Adapter Set 4 Operate the TissueLyser II for 2 min at 25 Hz Optional If working with fiber rich tissue disassemble the adapter set rotate the rack of tubes so that the tubes nearest to the TissueLyser ll are now outermost and reassemble the adapter set Operate the TissueLyser II
14. months at room temperature 15 25 C Procedure 1 Add up to 1 5 ml fluid sample to the Pathogen Lysis tube and centrifuge the tube for 5 min at maximum speed gt 14 000 x g 2 Remove and discard the supernatant Repeat steps 1 and 2 if necessary Use a pipet to remove the supernatant being careful not to remove any glass beads 3 Add 500 pl Buffer ATL containing Reagent DX and resuspend the pellet 4 Place the Pathogen Lysis Tube on a vortexer with a Microtube Foam Insert and vortex for 10 min at maximum speed Alternatively the Pathogen Lysis Tube may be processed on a TissueLyser LT for 10 min at 50 Hz or on a FastPrep 24 by applying a velocity of 6 5 m s for two 45 s periods with a 5 min resting time between them 5 Remove the Pathogen Lysis Tube from the vortexer and briefly centrifuge the tube to remove drops from the inside of the lid Use 200 ul of the supernatant as starting material for the protocol Purification of Pathogen Nucleic acids from Fluid Samples page 20 24 QlAamp cador Pathogen Mini Handbook 06 2012 Pretreatment B3 for Easy to Lyse Bacteria in High Volume Cell Free Fluids This optional pretreatment is designed to increase sensitivity using a high sample volume for the extraction of bacterial DNA of easy to lyse bacteria from cell free fluids such as serum Procedure 1 Add up to 1 5 ml fluid sample to a 2 ml microcentrifuge tube and centrifuge for 5 min at maximum speed gt 14 0
15. proteinase K into a 2 ml microcentrifuge tube not provided 2 Add 200 pl fluid sample to the proteinase K Note If processing lower sample volumes adjust the volume to 200 ul with PBS or 0 9 NaCl 3 Add 100 ul Buffer VXL Close the cap and mix by pulse vortexing To ensure sufficient lysis thoroughly mix the sample and Buffer VXL to yield a homogenous solution If using sample fluid containing Buffer ATL e g after enzymatic digestion of tissue precipitates may form Precipitates can be dissolved by brief incubation at 56 C However they have no influence on subsequent protocol steps Note If processing cell free samples ensure that 1 Ug Carrier RNA is added per 100 ul of Buffer VXL before use Do not add Carrier RNA if processing cell rich samples such as whole blood and tissue 4 Incubate at 20 25 C for 15 min 5 Briefly centrifuge the 2 ml tube to remove drops from the inside of the lid 6 Add 350 ul Buffer ACB to the sample close the cap and mix thoroughly by pulse vortexing Ensure that isopropanol was added to the Buffer ACB concentrate before use 7 Briefly centrifuge the 2 ml tube to remove drops from inside the lid 8 Transfer the lysate from step 7 to the QlAamp Mini column placed in a 2 ml collection tube without wetting the rim Close the cap and centrifuge at 6000 x g 8000 rpm for 1 min Place the QlAamp Mini column into a clean 2 ml collection tube and discard the collection tube containing the
16. to lyse bacteria from fluid samples or pretreated tissue samples The sample volumes can be up to 200 ul Important points before starting E Before beginning the procedure read Important Notes page 14 M Check that Buffer ACB Buffer AW1 and Buffer AW2 and carrier RNA have been prepared according to the instructions in Preparing reagents page 17 Hi Check that Buffer VXL or Buffer ACB does not contain a precipitate If necessary incubate Buffer VXL or ACB for 30 minutes at 37 C with occasional shaking to dissolve precipitate Things to do before starting M If necessary thaw and equilibrate the samples at room temperature 15 25 C M If the volume of the samples is less than 200 ul add PBS or 0 9 NaCl to a final volume of 200 ul M f necessary prepare a mixture of Buffer VXL and carrier RNA according to Table 3 for use in step 3 of the procedure Note Prepare a volume of the Buffer VXL Carrier RNA internal control mixture that is 10 greater than that required for the total number of sample purifications to be performed Table 5 Preparation of a Buffer VXL carrier RNA mixture Number of samples Reagent 1 12 30 Buffer VXL 100 ul 1 32 ml 3 3 ml Carrier RNA 1 ug ul 1 ul 13 ul 33 ul The volume prepared is 110 of the required volume to compensate for pipetting error and possible evaporation AAA a RI E 20 QlAamp cador Pathogen Mini Handbook 06 2012 Procedure 1 Pipet 20 ul
17. 00 x g 2 Remove and discard the supernatant Repeat steps 1 and 2 if necessary 3 Resuspend the pellet in 200 ul PBS by vigorous vortexing Use 200 ul from step 3 as starting material for the protocol Purification of Pathogen Nucleic acids from Fluid Samples page 20 AAPP EG QlAamp cador Pathogen Mini Handbook 06 2012 25 Pretreatment T1 Mechanical Disruption of Tissue This pretreatment is for the extraction of viral RNA and viral DNA from most types of tissue It is not suitable for bacterial DNA due to the centrifugation step Important point before starting M Stainless steel beads must be ordered separately for ordering information see page 36 Procedure 1 Place up to 25 mg tissue in 2 ml microcentrifuge tubes each containing 1 stainless steel bead 5 mm mean diameter For tissues with a very high number of cells for a given mass of tissue e g spleen a reduced amount of starting material 5 10 mg should be used If working with fibrous tissue cutting the tissue into smaller pieces before starting disruption will improve disruption efficiency 2 Add 300 pl PBS or 0 9 NaCl solution to each tube Place the tubes in the TissueLyser II Adapter Set 4 Operate the TissueLyser Il for 2 min at 25 Hz Optional If working with fiber rich tissue disassemble the adapter set rotate the rack of tubes so that the tubes nearest to the TissueLyser II are now outermost and reassemble the adapter set Operate
18. 100 ul of Buffer VXL containing dissolved carrier RNA is used per prep Note Carrier RNA does not dissolve in Buffer VXL It must first be dissolved in Buffer AVE The Buffer VXL solution containing dissolved carrier RNA should be prepared fresh and is stable at room temperature 15 25 C for up to 48 hours QlAamp cador Pathogen Mini Handbook 06 2012 17 QIAGEN Proteinase K The QlAamp cador Pathogen Mini Kit contains ready to use proteinase K supplied in a specially formulated storage buffer The activity of the proteinase K solution is 600 mAU ml QIAGEN Proteinase K is stable for at least 1 year after delivery when stored at room temperature 15 25 C To store for more than 1 year or if ambient temperature often exceeds 25 C we recommend storing proteinase K at 2 8 C Buffer ACB Buffer ACB is supplied as a concentrate Before using for the first time the appropriate amount of isopropanol 100 must be added as indicated on the bottle and in Table 2 Tick the check box on the bottle label to indicate that isopropanol has been added Mix well after adding isopropanol Table 2 Preparation of Buffer ACB No of preps ACB concentrate Isopropanol Final volume 50 12 ml 8ml 20 ml 250 60 ml 40 ml 100 ml Buffer AW1 Buffer AW1 is supplied as a concentrate Before using for the first time the appropriate amount of ethanol 96 100 must be added to Buffer AW1 as indicated on the bottle and in Table 3 Tick
19. June 2012 QlAamp cador Pathogen Mini Handbook For the purification of viral RNA and DNA and bacterial DNA from animal whole blood serum plasma other body fluids swabs and washes and tissue 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 6 Introduction 7 Principle and procedure 7 Description of protocols 7 Nucleic acid purification protocol 9 Pretreatments 9 Equipment and Reagents to Be Supplied by User 12 Important Notes 14 Protocols E Purification of Pathogen Nucleic Acids from Fluid Samples 20 a Pretreatment B1 for Difficult to Lyse Bacteria in Whole Blood or Pretreated Tissue 23 a Pretreatment B2 for Difficult to Lyse Bacteria in Cell Free Fluids 24 a Pretreatment B3 for Easy to Lyse Bacteria in High Volume Cell Free Fluids 25 a Pretreatment T1
20. Mini column After addition of Buffer AVE the QlAamp Mini not incubated with column should be incubated at room Buffer AVE before temperature 15 25 C for 1 min elution 32 QlAamp cador Pathogen Mini Handbook 06 2012 Comments and suggestions e Carrier RNA not added For samples containing a low number of cells to Buffer VXL not using carrier RNA may decrease the recovery of pathogen nucleic acids For these samples reconstitute carrier RNA in Buffer AVE and add reconstituted carrier RNA to Buffer VXL as described on page 17 Repeat the purification procedure with new samples f Degraded carrier RNA Carrier RNA reconstituted in Buffer AVE was not stored at 20 C or underwent multiple freeze thaw cycles Alternatively Buffer VXL carrier RNA mixture was stored for more than 48 h at 2 8 C Prepare a new tube of carrier RNA dissolved in Buffer AVE and mix with Buffer AL Repeat the purification procedure with new samples g Buffer VXL carrier RNA Mix Buffer AL with carrier RNA by gently inverting mixture mixed the tube of Buffer AL carrier RNA at least 10 times insufficiently h RNase contamination If tubes containing Buffer AVE are accessed in Buffer AVE repeatedly be careful to not introduce RNases which can degrade viral RNA In case of RNase contamination replace the open vial of Buffer AVE with a new vial Repeat the purification procedure with new samples i Nucleic acids in Samples were frozen and thawed more tha
21. ation with the TissueLyser LT Adapter 12 Tube 36 QlAamp cador Pathogen Mini Handbook 06 2012 Product Contents Cat no Internal Control For approximately 200 sample preps 211492 RNA High conc depending on elution volume Lyophilized Internal Control RNA Nucleic Acid Dilution Buffer Internal Control For approximately 200 sample preps 211392 DNA High Conc depending on elution volume Lyophilized Internal Control RNA Nucleic Acid Dilution Buffer QuantiFast For 100 x 25 ul reactions Master Mix 211352 Pathogen PCR IC lyophilized Internal Control Assay Kit 100 lyophilized Internal Control DNA ROX Dye Solution High ROX Dye Solution RNase Free Water Nucleic Acid Dilution Buffer Buffer TE QuantiFast For 100 x 25 ul reactions Master Mix RT 211452 Pathogen RT PCR Mix lyophilized Internal Control Assay IC Kit 100 lyophilized Internal Control RNA ROX Dye Solution High ROX Dye Solution RNase Free Water Nucleic Acid Dilution Buffer Buffer TE 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 qiagen com or can be requested from QIAGEN Technical Services or your local distributor QlAamp cador Pathogen Mini Handbook 06 2012 37 Notes 38 QlAamp cador Pathogen Mini Handbook 06 2012 Trademarks QIAGEN QlAamp cador QIAGEN Group FastPrep MP Biomedica
22. e drying step step 11 in the protocol eluate Purification of Pathogen Nucleic acids from Fluid Samples page 20 Precipitate in buffers a Precipitate in Buffer VXL Precipitate may form after storage at low or Buffer ACB temperature or prolonged storage To dissolve precipitate incubate Buffer VXL or ACB for 30 min at 37 C with occasional shaking b Precipitate in sample If using sample fluid containing Buffer ATL e g Buffer VXL mixture after enzymatic digestion of tissue precipitate may form after addition of Buffer VXL to the sample step 3 of the protocol Purification of Pathogen Nucleic acids from Fluid Samples The precipitate does not influence subsequent protocol steps However the precipitate can be dissolved by brief incubation at 56 C 34 QlAamp cador Pathogen Mini Handbook 06 2012 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 giagen com RefDB search asp or contact QIAGEN Technical Services or your local distributor AAA A A A A AAA A QlAamp cador Pathogen Mini Handbook 06 2012 35 Ordering Information Product Contents Cat no QlAamp cador For 50 preps QlAamp Mini C
23. for a further 2 min at 25 Hz 5 Disassemble the adapter set Place the tubes containing the homogenates on the bench top at room temperature 15 25 C for 2 3 min This step promotes dissociation of nucleoprotein complexes 6 Add 100 pl chloroform Securely cap each tube containing the homogenate and shake vigorously for 15 s by vortexing or repeated inversion of the tube Thorough mixing is important for subsequent phase separation 30 QlAamp cador Pathogen Mini Handbook 06 2012 7 Place the tube containing the homogenate on the bench top at room temperature for 3 min 8 Centrifuge at 12 000 x g for 15 min at 4 C After centrifugation the sample separates into 3 phases an upper colorless aqueous phase containing nucleic acids a white interphase and a lower organic phase For tissues with an especially high fat content an additional clear phase may be visible below the organic phase The volume of the aqueous phase should be approximately 300 ul Important If the same centrifuge is to be used the protocol Purification of Pathogen Nucleic acids from Fluid Samples page 20 heat the centrifuge to room temperature 9 Use 200 pl of the upper phase from step 7 as starting material for the protocol Purification of Pathogen Nucleic acids from Fluid Samples page 20 AP aaa QlAamp cador Pathogen Mini Handbook 06 2012 31 Troubleshooting Guide This troubleshooting guide may be helpful in solving any
24. in cell free fluids E Vortexer with Microtube foam insert Scientific Industries cat no 504 0234 00 or TurboMix Attachment Scientific Industries cat no SI 0564 or FastPrep 24 MP Biomedicals cat no 6004500 or TissueLyser cat no 85300 with a TissueLyser ll Adapter Set 2 x 24 cat no 69982 or 2 x 96 cat no 69984 or TissueLyser LT cat no 85600 with the TissueLyser LT Adapter for 12 tubes cat no 69980 M Pathogen Lysis Tubes L cat no 19092 or S cat no 19091 containing 50 Pathogen Lysis Tubes with glass beads and 1 vial Reagent DX cat no 19088 for bead beating of bacteria E Buffer ATL cat no 19076 Pretreatment TI mechanical disruption of tissue M Tissuelyser II cat no 85300 with a TissueLyser ll Adapter Set 2 x 24 cat no 69982 or TissueLyser LT cat no 85600 with the TissueLyser LT Adapter for 12 tubes cat no 69980 or other bead mill homogenizer Note A Vortexer with microtube foam insert Scientific Industries cat no 504 0234 00 can be used instead E 5 mm stainless steel beads cat no 69989 E PBS pH 7 2 50 mM potassium phosphate 150 mM NaCl or NaCl 0 9 Pretreatment T2 enzymatic digestion of tissue BE Thermoshaker suitable for 2 ml collection tubes E Buffer ATL cat no 19076 This is not a complete list of suppliers and does not include many important vendors of biological supplies QlAamp cador Pathogen Mini Handbook 06 2012 13 Important
25. ls LLC Limited License Agreement for QlAamp cador Pathogen Mini Kit Use of this product signifies the agreement of any purchaser or user of the product to the following terms 1 The product may be used solely in accordance with the protocols provided with the product and this 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 except as described in the protocols provided with the product this handbook and additional protocols available at www giagen com Some of these additional protocols have been provided by QIAGEN users for QIAGEN users These protocols have not been thoroughly tested or optimized by QIAGEN QIAGEN neither guarantees them nor warrants that they do not infringe the rights of third parties 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 Or BN 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
26. mains in the tubes after lysis add 50 pl Buffer ATL Mix by vortexing and centrifuge at 6 000 x g 8 000 rpm for 1 min Use 200 ul of the supernatant in step 5 5 Use 200 pl lysate as starting material for step 5a or 5b Important Ensure that no solid particles are transferred to the next protocol 5a For isolation of viral DNA or DNA from easy to lyse bacteria proceed directly with the protocol Purification of Pathogen Nucleic Acids from Fluid Samples page 20 Note As mentioned above do not use proteinase K in step 1 of the purification protocol 5b For isolation of DNA from difficult to lyse bacteria proceed with Pretreatment B1 page 23 28 QlAamp cador Pathogen Mini Handbook 06 2012 Pretreatment T3 Rapid Partial Disruption of Tissue This pretreatment is suitable for applications that do not require complete tissue disruption It is for the extraction of viral RNA and DNA and bacterial DNA from tissues Important points before starting For some tissues and pathogens vigorous shaking is sufficient to disassemble cells and or release pathogens from the tissue structure Typically released cells will not be disrupted by this pretreatment Cell lysis is performed subsequently in the protocol Purification of Pathogen Nucleic acids from Fluid Samples page 20 The suitability of this protocol depends on the strength of cell adhesion and or pathogen location within the tissue and on the required sensitivit
27. me is flexible and can be adapted according to requirements of the downstream application To reduce noise the centrifugation speed for elution can be set to 6000 x g If this is done the recovered eluate volume will be approximately 5 ul less than elution buffer volume applied onto the column 22 QlAamp cador Pathogen Mini Handbook 06 2012 Pretreatment B1 for Difficult to Lyse Bacteria in Whole Blood or Pretreated Tissue This pretreatment is for the extraction of DNA of difficult to lyse bacteria from whole blood or pretreated tissue Important points before starting E Buffer ATL and Pathogen Lysis Tubes L with glass beads including Reagent DX must be ordered separately for ordering information see page 36 E Buffer ATL may form precipitates upon storage If necessary warm to 56 C until the precipitates have fully dissolved Things to do before starting E Before use add 100 ul Reagent DX to 15 ml Buffer ATL If smaller amounts are needed transfer 1 5 ml of Buffer ATL into a sterile 2 ml vial and add 10 ul Reagent DX Mix well after addition of Reagent DX After preparation the mixture is stable for 6 months at room temperature 15 25 C Procedure 1 Add 100 ul Buffer ATL containing Reagent DX into a fresh Pathogen Lysis Tube 2 Add 400 ul blood or other sample fluids If using less starting material adjust the volume to 400 ul with PBS or 0 9 NaCl 3 Place the Pathogen Lysis Tube on a vortexer with a
28. n samples already once or stored at room temperature for too long degraded prior to Always use fresh samples or samples thawed purification only once Repeat the purification protocol with new samples DNA or RNA does not perform well in downstream applications a Little or no DNA or See Little or no pathogen DNA or RNA in the RNA in the eluate eluate above for possible reasons QlAamp cador Pathogen Mini Handbook 06 2012 33 Comments and suggestions b Too much eluate in the Some sample types may contain high amounts of amplification reaction background nucleic acids e g animal whole blood tissue or PCR inhibiting substances feces High amounts of background nucleic acids may inhibit amplification reactions and removal of inhibitors might not be complete without special treatment Reduce the amount of sample input or and the amount of eluate added to the amplification reaction c Too much carrier RNA Determine the maximum amount of carrier RNA in the eluate suitable for your amplification reaction Adjust the concentration of carrier RNA solution added to the Buffer VXL accordingly d Performance of purified Salt and ethanol components of Buffer AW nucleic acids in assays downstream or Buffer AW2 may have separated varies with aging of out after being left for a long period between reconstituted wash preparations Always mix buffers thoroughly buffers before each preparation e Residual ethanol inthe Use th
29. olumns 54104 Pathogen Mini Kit QIAGEN Proteinase K Carrier RNA 50 Buffers Collection Tubes 2 ml QlAamp cador For 250 preps QlAamp Mini Columns 54106 Pathogen Mini Kit QIAGEN Proteinase K Carrier RNA 250 Buffers Collection Tubes 2 ml Buffer ATL 200 ml 200 ml Tissue Lysis Buffer for 1000 preps 19076 TissueLyser II Bead mill 100 120 220 240 V 50 60 Hz 85300 requires the TissueLyser Adapter Set 2 x 24 or TissueLyser Adapter Set 2 x 96 available separately TissueLyser Adapter 2 sets of adapter plates and 2 racks for 69982 Set 2 x 24 use with 2 ml microcentrifuge tubes on the TissueLyser II TissueLyser Adapter 2 sets of adapter plates for use with 69984 Set 2 x 96 Collection Microtubes racked on the TissueLyser II TissueLyser LT Compact bead mill 100 240 V AC 85600 50 60 Hz requires the TissueLyser LT Adapter 12 Tube available separately TissueLyser LT Adapter for disruption of up to 12 69980 Adapter 12 Tube samples in 2 ml microcentrifuge tubes on the TissueLyser LT Pathogen Lysis 50 Pathogen Lysis Tubes and 1 vial of 19092 Tubes L Reagent DX Pathogen Lysis 50 Pathogen Lysis Tubes and 1 vial of 19091 Tubes S Reagent DX Stainless Steel 200 stainless steel beads 5 mm 69989 Beads 5 mm 200 diameter suitable for use with TissueLyser systems The TissueLyser ll must be used in combination with the TissueLyser Adapter Set 2 x 24 or TissueLyser Adapter Set 2 x 96 t The TissueLyser LT must be used in combin
30. ommend using 50 200 ul blood per sample Typically 200 ul of blood can be used with most blood samples However highly elevated cell counts due to inflammatory or neoplastic diseases may strongly increase the host nucleic acid content of a sample In this case reduction of sample input to 50 ul may improve results in downstream assays particularly in RT PCR If using less than 200 ul blood adjust the sample volume to 200 ul with PBS or 0 9 NaCl For blood samples containing nucleated erythrocytes e g samples from bird and fish use 5 25 pl blood and adjust the sample volume to 200 pl with PBS or 0 9 NaCl 14 QlAamp cador Pathogen Mini Handbook 06 2012 Animal serum plasma other body fluids swab and wash specimens Frozen plasma or serum must not be thawed more than once before processing We recommend storing swabs in transport media for example viral transport media VTM or brain heart infusion broth BHI Remove the swab and squeeze out the liquid by pressing the swab against the inside of the storage tube For extraction of viral RNA or DNA we recommend centrifuging the swab media briefly to ensure any residual solid materials are removed Note Solid pieces remaining in the sample fluid may aggregate on the QlAamp Mini column which may decrease nucleic acid yield Up to 200 ul serum plasma other body fluid swab media supernatant or wash fluid can be processed Carrier RNA must be used in the nucleic acid purificati
31. on protocol to prevent the loss of nucleic acids during the procedure see page 16 for information on the use of carrier RNA The processing of samples with very high inhibitor contents such as urine or feces suspensions may require a reduction in sample input volume and or an extra pretreatment to remove inhibitors To reduce the input volume use 25 50 ul of the sample and adjust the volume to 200 ul with PBS or 0 9 NaCl For extraction of bacterial DNA the input volume can be increased to more than 200 ul e g 1 5 ml for increased sensitivity of bacterial detection See Pretreatment B2 page 24 for extraction of DNA from difficult to lyse bacteria and Pretreatment B3 page 25 for extraction of DNA from easy to lyse bacteria Animal tissues When working with tissue samples mechanical or enzymatic disruption of the tissue structure is the prerequisite for liberation of cells subsequent release of nucleic acids and membrane permeability of the material Different tissue types can vary widely with regard to texture and rigidity cell types and content of host nucleic acids and inhibitory substances In addition the localization of pathogen nucleic acids in the tissue may vary depending on tissue type pathogen and stage of infection Therefore suitability of the pretreatment protocols in this handbook should be evaluated for each new combination of tissue and pathogen Up to 25 mg of fresh or frozen tissue can be used as a star
32. 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 giagen com Comments and suggestions Little or no pathogen DNA or RNA in the eluate a Buffer ACB prepared Check that Buffer ACB concentrate was diluted incorrectly with the correct volume of isopropanol as indicated on the bottle Use 100 isopropanol Repeat the purification protocol with new samples b Buffer AW1 or Buffer Check that Buffer AW1 or Buffer AW2 AW prepared concentrate was diluted with the correct volume incorrectly of ethanol as indicated on the bottle Use 96 100 ethanol Do not use denatured alcohol which contains other substances such as methanol or methylethylketone Repeat the purification protocol with new samples c Insufficient sample lysis Proteinase K was stored at elevated temperatures for too long Repeat the purification procedure using new samples and fresh proteinase K see storage recommendations on page 5 For some DNA viruses and bacteria heated lysis may improve lysis efficiency In this case mix the sample thoroughly after addition of proteinase K and incubate for 15 min at 70 C d QlAamp
33. rgents in the lysis buffer Not using carrier RNA may decrease the recovery of pathogen nucleic acids Do not add carrier RNA to whole blood and tissue samples or other samples containing a high amount of cells Internal control Use of an internal control such as the QIAGEN Internal Control to be used with QuantiFast Pathogen IC Kits see page 37 for ordering information is optional depending on the amplification system used Using the QlAamp cador Pathogen Mini Kit in combination with amplification systems that use an internal control may require introduction of these internal controls during the purification procedure to monitor the efficiency of sample preparation and downstream assay Add unprotected internal control nucleic acids e g plasmid DNA or in vitro transcribed RNA to the sample lysate or Buffer VXL only Do not add these internal control nucleic acids directly to the sample The amount of internal control added depends on the assay system and the elution volume Evaluation of the correct amount of internal control nucleic acid must be performed by the user Refer to the manufacturer s instructions to determine the optimal concentration of internal control 16 QlAamp cador Pathogen Mini Handbook 06 2012 Storing nucleic acids For short term storage of up to 24 hours we recommend storing the purified viral RNA and DNA or bacterial DNA at 2 8 C For storage longer than 24 hours we recommend storing purified nucleic
34. rrier RNA can be stored at room temperature until the expiration date stated on the kit box For use lyophilized carrier RNA should be dissolved in Buffer AVE and then added to Buffer VXL as described in Preparing reagents on page 17 This carrier RNA Buffer AVE Buffer VXL solution should be prepared fresh and is stable at room temperature for up to 48 hours Unused carrier RNA dissolved in Buffer AVE should be immediately frozen in aliquots at 20 C Do not subject aliquots of carrier RNA to more than 3 freeze thaw cycles QIAGEN Proteinase K can be stored at room temperature To store for extended periods of time or if the ambient temperature often exceeds 25 C we recommend storing at 2 8 C Intended Use The QlAamp cador Pathogen Mini Kit is intended for the extraction of pathogen nucleic acids viral RNA and DNA and bacterial DNA from animal whole blood serum plasma other body fluids swabs washes and tissue For laboratory use Not for use in veterinary diagnostic procedures No claim or representation is intended to provide information for the diagnosis prevention or treatment of a veterinary 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 QlAamp cador Pathogen Mini Handbook 06 2012 5 Safety Information
35. s always wear a suitable lab coat disposable gloves and protective goggles For more information consult the appropriate safety data sheets SDSs available from the product supplier For all protocols Pipettors and disposable pipet tips with aerosol barriers 20 1000 ul Isopropanol Ethanol 96 100 Phosphate buffered saline PBS may be required for sample dilution Microcentrifuge 2 ml microcentrifuge tubes Vortexer Pretreatment B1 for difficult to lyse bacteria in whole blood or pretreated tissue E Vortexer with Microtube foam insert Scientific Industries cat no 504 0234 00 or TurboMix Attachment Scientific Industries cat no SI 0564 or FastPrep 24 MP Biomedicals cat no 6004500 or TissueLyser II cat no 85300 with a TissueLyser ll Adapter Set 2 x 24 cat no 69982 or 2 x 96 cat no 69984 or TissueLyser LT cat no 85600 with the TissueLyser LT Adapter for 12 tubes cat no 69980 M Pathogen Lysis Tubes L cat no 19092 containing 50 Pathogen Lysis Tubes with glass beads and 1 vial Reagent DX cat no 19088 for bead beating of bacteria E Buffer ATL cat no 19076 Denatured alcohol is not suitable as it contains other substances such as methanol or methylethylketone t This is not a complete list of suppliers and does not include many important vendors of biological supplies 12 QlAamp cador Pathogen Mini Handbook 06 2012 Pretreatment B2 for difficult to lyse bacteria
36. the TissueLyser II for a further 2 min at 25 Hz Pd 5 Disassemble the adapter set Centrifuge the samples at 14 000 x g for 2 min at room temperature 15 25 C 6 Use 200 ul of the supernatant from step 5 as the starting material for the protocol Purification of Pathogen Nucleic acids from Fluid Samples page 20 For fiber rich tissues complete disruption may not always be possible Ensure that no solid particles are transferred to the purification protocol 26 QlAamp cador Pathogen Mini Handbook 06 2012 Pretreatment T2 Enzymatic Digestion of Tissue This pretreatment is for the extraction of bacterial and viral DNA from most types of tissue It is not suitable for viral RNA because the lysis conditions do not sufficiently conserve RNA integrity Important point before starting E Buffer ATL must be ordered separately for ordering information see page 36 Things to do before starting E Buffer ATL may form precipitates upon storage If necessary warm to 56 C until the precipitates have fully dissolved E Heat a thermomixer block shaking water bath or rocking platform to 56 C for use in step 3 of the pretreatment protocol Procedure 1 Cut up to 25 mg tissue into small pieces and place in a 2 ml microcentrifuge tube Add 180 ul Buffer ATL For tissues with a very high number of cells for a given mass of tissue e g spleen a reduced amount of starting material 5 10 mg should be used We recommend cut
37. the check box on the bottle label to indicate that ethanol has been added Mix well after adding ethanol Table 3 Preparation of Buffer AW1 No of preps AW1 concentrate Ethanol Final volume 50 19 ml 25 ml 44 ml 250 98 ml 130 ml 228 ml QlAamp cador Pathogen Mini Handbook 06 2012 Buffer AW2 Buffer AW2 is supplied as a concentrate Before using for the first time the appropriate amount of ethanol 96 100 must be added as indicated on the bottle and in Table 4 Tick the check box on the bottle label to indicate that ethanol has been added Mix well after adding ethanol Table 4 Preparation of Buffer AW2 No of preps AW2 concentrate Ethanol Final volume 50 17 ml 40 ml 57 ml 250 81 ml 190 ml 271 ml Handling Buffer AVE Buffer AVE is RNase free upon delivery lt contains sodium azide an antimicrobial agent that prevents growth of RNase producing organisms However as this buffer does not contain any RNase degrading chemicals it will not actively inhibit RNases introduced by inappropriate handling For RNA applications when handling Buffer AVE avoid contamination with RNases Follow general precautions for working with RNA such as frequent change of gloves and keeping tubes closed whenever possible QlAamp cador Pathogen Mini Handbook 06 2012 19 Protocol Purification of Pathogen Nucleic Acids from Fluid Samples This protocol is for the purification of viral RNA and DNA and the DNA of easy
38. ting amount For tissues with a very high number of cells for a given mass of tissue such as spleen a reduced amount of starting material 5 10 mg should be used QlAamp cador Pathogen Mini Handbook 06 2012 15 Yields of nucleic acids For samples containing a low amount of cells e g serum the yield of viral or bacterial nucleic acids obtained can be below 1 ug and is therefore difficult to quantify using a spectrophotometer In addition eluates prepared with carrier RNA may contain much more carrier RNA than target nucleic acids The QlAamp cador Pathogen Mini protocol recovers total nucleic acids Therefore cellular DNA and RNA will be copurified from any cells in the sample along with viral RNA and DNA and bacterial DNA and cannot be distinguished using spectrophotometric measurements We recommend using quantitative amplification methods such as quantitative real time PCR or real time RT PCR to determine pathogen nucleic acid yields Using carrier RNA and internal controls Carrier RNA We recommend adding carrier RNA to fluids containing a low amount of cells such as serum plasma swabs media and wash fluid as it enhances adsorption of viral RNA and DNA and bacterial DNA to the silica membranes which is especially important when the target molecules are not abundant In addition an excess of carrier RNA reduces the chances of viral RNA degradation in the rare event that RNases are not denatured by the chaotropic salts and dete
39. ting the tissue into small pieces for efficient lysis 2 Add 20 ul proteinase K Close the cap and mix thoroughly by vortexing Briefly centrifuge the tube to collect any solution from the cap Note When using this pretreatment do not use proteinase K in step 1 of the subsequent nucleic acid purification protocol Purification of Pathogen Nucleic acids from Fluid Samples on page 20 This digestion eliminates the need for the use of this reagent in that later step QlAamp cador Pathogen Mini Handbook 06 2012 27 3 Incubate at 56 C with constant agitation until the tissue is completely lysed Lysis time varies depending on the type of tissue processed Lysis is usually complete in 1 3 h If more convenient overnight lysis is possible but should be evaluated for specific sample types After incubation the lysate may appear viscous but should not be gelatinous If a substantial gelatinous pellet remains after incubation and vortexing extend incubation time at 56 C for proteinase K digest and or increase amount of proteinase K to 40 ul Reduce the amount of starting material in future preparations of this tissue type If no thermomixer shaking water bath or rocking platform is available incubate in a heating block or water bath and vortex occasionally during incubation to disperse the sample 4 Optional for viral DNA or DNA of easy to lyse bacteria not suitable for difficult to lyse bacteria If solid tissue or debris re
40. y of pathogen detection and must be evaluated for each new combination of tissue and pathogen Procedure 1 Place up to 25 mg tissue in a 2 ml microcentrifuge tube 2 Add 500 ul PBS to the sample and close the lid If a larger amount of tissue is used adapt the volume of PBS and size of tube accordingly Keep a ratio of approximately 1 20 m v for tissue and PBS Ensure that the tube volume is sufficient to allow vigorous shaking 3 Place the tube onto a vortexer fixed in a foam adapter in vertical or horizontal orientation 4 Vortex for 5 min at full speed Proceed with step 5 Important Do not allow the sample to stand for more than 1 min after vortexing If the sample has stood for more than 1 min agitate again by vortexing for a few seconds 5 Use 200 ul of the supernatant as starting material for the protocol Purification of Pathogen Nucleic acids from Fluid Samples page 20 Ensure that no solid particles are transferred to the purification protocol 5a For isolation of viral DNA or DNA from easy to lyse bacteria proceed directly with the protocol Purification of Pathogen Nucleic Acids from Fluid Samples page 20 5b For isolation of DNA from difficult to lyse bacteria proceed with Pretreatment B1 page 23 QlAamp cador Pathogen Mini Handbook 06 2012 29 Pretreatment T4 Organic Extraction for Difficult Tissue This pretreatment is for the extraction of viral RNA and DNA and DNA of easy to
41. y result in a substantial loss of bacterial DNA with the pelleted material To extract bacterial or viral DNA from tissue use Pretreatment T2 page 27 This pretreatment is not suitable for the extraction of viral RNA because the lysis conditions do not sufficiently conserve RNA integrity As mentioned on page 9 depending on the bacteria an additional pretreatment may be needed after Pretreatment T2 To extract the DNA of difficult to lyse bacteria from tissue after Pretreatment T2 use Pretreatment B1 page 23 10 QlAamp cador Pathogen Mini Handbook 06 2012 For some applications partial tissue disruption by vigorous shaking may be sufficient to release an adequate amount of cells from the tissue structure for reliable pathogen identification This can be the case for tissue with weak cell adhesion for pathogens located in the extracellular space or in easily detached cells or for applications with high pathogen loads In this case Pretreatment T3 page 29 may be suitable For difficult to lyse bacteria subsequently use Pretreatment B1 page 23 Some tissue types such as pancreas or brain tissue can be very difficult to process due to their high lipid and or nuclease content To extract viral RNA and DNA and bacterial DNA of easy to lyse bacteria from these tissues use Pretreatment T4 page 30 AAPP QlAamp cador Pathogen Mini Handbook 06 2012 11 Equipment and Reagents to Be Supplied by User When working with chemical
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