GeneJET™ Genomic DNA Purification Kit
Contents
1. reconstitute the dried Carrier RNA by adding 300 pl of Eluent Allow the freshly reconstituted Carrier RNA to incubate for 5 min at room temperature then mix thoroughly and briefly centrifuge the vial Use immediately or store at 20 C Do not freeze thaw the reconstituted Carrier RNA more than 10 times If only few samples will be processed at a time divide the Carrier RNA solution into 50 ul aliquots using nuclease free tubes and store at 20 C or 70 C CALCULATING THE REQUIRED QUANTITY OF CARRIER RNA When starting a new procedure always use a freshly prepared mixture of Carrier RNA and Lysis Solution To calculate the correct quantity of Carrier RNA and Lysis Solution required to process multiple samples use the following table Supplement Lysis Solution with the required quantity of Carrier RNA and mix by pulse vortexing or pipetting No Vol Lysis Vol Carrier No Vol Lysis Vol Carrier samples Solution ml RNA ul samples Solution ml RNA ul 1 0 22 5 5 13 2 86 71 5 2 0 44 11 0 14 3 08 71 0 3 0 66 16 5 15 3 30 82 5 4 0 88 22 0 16 3 52 88 0 5 1 10 2 17 3 74 93 5 6 1 32 33 0 18 3 96 99 0 7 1 54 38 5 19 4 18 104 5 8 1 76 44 0 20 4 40 110 0 9 1 98 49 5 21 4 62 115 5 10 2 20 55 0 22 4 84 121 0 11 2 42 60 5 23 5 06 126 5 12 2 64 66 0 24 5 28 132 0 Note Required volumes ul of Carrier RNA are calculated using following formula N x 0 22 ml Y ml Y ml x 25 0 ul ml Z ul Where N number of samples to be proces2. Use sterile disposable RNase free pipette tips Remove RNase contamination from non disposable items and work surfaces Poor quality of samples Always use fresh samples or samples handled as recommended on page 4 For lysis process the sample quickly to avoid degradation 11 SAFETY INFORMATION Lysis Solution Xn Harmful Hazard determining component of labeling Guanidinium chloride Risk phrases R22 Harmful if swallowed R38 Irritating to skin R41 Risk of serious damage to eyes R 52 53 Harmful to aquatic organisms may cause long term adverse effects in the aquatic environment Safety phrases S23 Do not breathe gas fumes vapour spray S26 In case of contact with eyes rinse immediately with plenty of water and seek medical advice S36 37 39 Wear suitable protective clothing gloves and eye face protection 60 This material and its container must be disposed of as hazardous waste 61 Avoid release to the environment Refer to special instructions safety data sheets Wash Buffer 1 Xn Harmful Hazard determining component of labeling Guanidinium chloride Risk phrases R22 Harmful if swallowed R 36 38 Irritating to eyes and skin Safety phrases 23 Do not breathe gas fumes vapour spray 26 In case of contact with eyes rinse immediately with plenty of water and seek medical advice 536 37 Wear suitable protective clothing and gloves 60 This material and its container must be disposed of a
3. purified viral nucleic acids are free of proteins nucleases and other contaminants or inhibitors of downstream applications Isolated DNA and RNA can be directly used in PCR qPCR or other nucleic acid based assays Host genomic DNA or RNA co purified from liquid cell containing samples typically does not interfere with viral nucleic acid yields due to high capacity of the spin column membrane up to 50 yg To minimize irregularities in results the product must be used with an appropriate internal control as well as positive and negative controls throughout the process of sample preparation amplification and detection PRINCIPLE The Gene ET Viral DNA and RNA Purification Kit uses well established nucleic acid isolation and purification technique comprised of following steps Sample lysis The sample is lysed by incubation with Lysis Solution and P roteinase K under l denaturating conditions at elevated temperatures 56 C The Lysis Solution and Proteinase K inactivate both RNases and DNases ensuring protection of viral nucleic acids against degradation Binding viral nucleic acids to the spin column membrane The lysed sample is transferred to a spin column where released viral nucleic acids immediately bind to the silica based filter in the presence of chaotropic salts The remaining lysate is removed by centrifugation Removing remaining contaminants 3 The remaining contaminants are removed during three wash steps using Wash Bu
4. PRODUCT INFORMATION Thermo Scientific Gene ET Viral DNA and RNA Purification Kit K0821 www thermoscientific com onebio Packaging Lot __ Exp __ CERTIFICATE OF ANALYSIS Quality of the kit was tested by purification of RNA and DNA from 200 ul of spiked human plasma Yield of nucleic acids was evaluated by RT gPCR and qPCR and compared with Spiked copy number Quality authorized by gx J urgita Zilinskiene Rev2 Ill 66 CONTENTS page COMPONENTS OF THE RIT isis qiie oes eibl tui ta reb o c id tete aaa mati 2 STORAG Earrainn AN Aaa ai 2 DESCRIPTIO Ningri ireira ce arden AN TENAR EA A ER tase 2 PRINCIPLE dba compenso oet Mathei at A wack EA E a 3 IMPORTANT NOTE Sinn st here Bc itd ean A TEAT a 3 SAMPLE HANDLING cod tira rta ad HD O Ode HD Co Gn e 4 PREPARING REAGENTS AND BUFFERS daz Dei bud He phe budi de ertet 5 CARRIER RNA ndis aiia rtt la noi hte tree tates Mettre ut teda 5 PREPARATION OF CARRIER R NR aciscte toritate o eet PUR RU 5 CALCULATING THE REQUIRED QUANTITY OF CARRIER RNA seeenemnne 6 INTERNAL CONTROL td itasse pt itin Er ed d ANAE RE 6 ADDITIONAL MATERIALS AND EQUIPMENT REQUIRED eennnmne 6 PROTOCOLS vot errr rete a cer ibt Hunde TO PER tree re A dn 7 TROUBLESHOOTING santet tits tna sutor nun aerarii areis etus ca ind 11 SAFETY INFORMATION itai atte ances DU dead to annuas nO GG 12 REFERENCE c 13 COMPONENTS OF THE KIT Gene E
5. T Viral DNA and RNA Purification Kit E Column P reparation Liquid red cap 2x14 ml Lysis Solution 12 ml Wash Buffer 1 concentrated 25 ml Wash Buffer 2 concentrated 11 ml Eluent white cap 3x 1 25 ml Proteinase K green cap 2 x 1 3 ml Carrier RNA dried blue cap 1 vial Spin Columns preassembled with Wash Tubes 50 Wash Tubes 2 ml 4x50 Handbook 1 contains guanidine hydrochloride guanidinium chloride STORAGE When the kit is delivered remove the Carrier RNA from the package and store in the original aluminum bag at 20 C Other components of the kit should be stored at room temperature 15 25 C All components are stable until the listed expiration date Wash Buffer 1 and Wash Buffer 2 are stable until the listed expiration date after addition of ethanol DESCRIPTION The Thermo Scientific GeneJ ET Viral DNA and RNA Purification Kit is designed for rapid and efficient purification of high quality viral nucleic acids from various human and animal liquid samples such as plasma serum whole blood saliva nasal and buccal swabs urine cells should be collected before purification urogenital swabs and milk The kit utilizes a silica based membrane technology in the form of a convenient spin column Nucleic acids from lysed samples bind to the column membrane while impurities are effectively removed during subsequent washing and centrifugation steps Finally ready to use nucleic acids are eluted from the column The
6. be containing flow through Place the Spin Column into a new 2 ml Wash Tube Centrifuge the column for 3 min at 16 000 x g Discard the Wash Tube containing remaining flow through Place the Spin Column into a new 1 5 ml elution tube Add 50 ul of Eluent preheated to 56 C to the center of Spin Column membrane Incubate for 2 min at room temperature Centrifuge the column for 1 min at 13 000 x g Discard the Spin Column Note e Lower volume of Eluent 30 40 ul can be used in order to concentrate eluted nucleic acids Larger elution volumes up to 100 ul can also be used but may result in dilution of the viral nucleic acid sample 10 Keep the elution tube containing pure viral nucleic acids Use the purified nucleic acids immediately or store at 20 C or 70 C Note e qPCR and RT qPCR inhibition might occur if lower than 50 ul volume of Eluent is used for elution e For further use in downstream qPCR applications use 1 10 ul of viral DNA per 25 ul reaction volume For reverse transcription RT use 1 10 ul of viral RNA per 20ul cDNA synthesis reaction volume Nucleic acid purification from nasal and buccal swabs Step Procedure To collect a sample scrape the swab 5 6 times againstthe inside cheek or nose Swirl the swab for 2 3 min in 200 ul of 1 x PBS or TE buffer CIS IPSI Go to step 1 ofthe standard Viral Nucleic Acid Purification P rotocol page 7 C Nucleic acid pu
7. ffer 1 and Wash Buffer 2 whereas pure nucleic acids remain bound to the membrane Elution of pure viral nucleic acids 4 Pure viral nucleic acids are released from the spin column filter using Eluent The purified nucleic acids are ready for subsequent use in downstream applications IMPORTANT NOTES e Ensure the integrity of the kit components upon the delivery Contact technical service or your local distributor in case of damage Do not use damaged kit components e The Lysis Solution and Wash Buffer 1 contain irritants Always wear gloves and follow standard safety precautions when handling these reagents For more information refer to SAFETY INFORMATION page 12 and Material Safety Data Sheets e All sample material and waste should be regarded as potentially infectious Wear the proper protection when handling samples and waste Avoid any skin or eye contact Work under laminar air flow conditions if possible until samples are lysed Disinfect all work surfaces thoroughly after the procedure Follow proper waste disposal guidelines as recommended by local authorities e The following steps should be taken in order to avoid cross contamination always change pipette tips between liquid transfers aerosol barrier pipette tips recommended open only one tube ata time use disposable gloves and discard if contaminated e Always use RNase free equipment e Use only a freshly prepared mixture of Carrier RNA and Lysis Solution when beginnin
8. g a new extraction procedure e Before beginning the procedure a new Spin Column must be prepared by adding 50 yl of Column P reparation Liquid into the center of the column membrane Do not centrifuge the column after addition of Column Preparation Liquid 3 SAMPLE HANDLING e f possible use only fresh sample material Plasma serum whole blood samples can be stored at 2 8 C for up to 24 hours or at 20 C or 70 C for long term storage Urogenital swabs can be stored at 2 8 C for up to 48 hours For longer term storage cells should be collected by centrifugation and stored at 20 C or 70 C Nasal and buccal swabs can be stored at 2 8 C for up to 48 hours Urine samples should be stored at 2 8 C for up to 12 hours with 0 5 M EDTA added to 50 mM final concentration or at 20 C or 70 C for long term storage cells should be collected by centrifugation For viral RNA purification it is recommended to collect cells by centrifugation immediately after sample collection e Do not freeze thaw samples more than once Equilibrate samples to room temperature 20 5 C before use Remove precipitates from plasma serum samples if any by centrifugation for 5 min at 3 000 x g Use EDTA or citrate treated plasma samples PREPARING REAGENTS AND BUFFERS Add the indicated volume of ethanol 96 10096 to the concentrated Wash Buffer 1 and concentrated Wash Buffer 2 prior to first use K 0821 for 50 preps Wash B
9. ion P rotocol page 7 10 TROUBLESHOOTING Problem Possible cause and solution Low nucleic Improper spin column preparation acid yield Make sure the Spin Column has been prepared properly by adding Column P reparation Liquid as described on page 5 Ethanol was not added to the lysate Make sure that ethanol was added to the lysate before applying the sample to the purification column Ethanol was not added to Wash Buffers 1 and 2 Make sure that ethanol was added to Wash Buffers 1 and 2 prior to the first use Low percentage ethanol used Use only 96 100 ethanol Do not use denatured or 95 ethanol Carrier RNA was not added to the lysate Reconstitute carrier RNA in Eluent and mix with Lysis Solution as described on page 5 Degraded carrier RNA Do not freeze thaw the reconstituted Carrier RNA more than 10 times Store at 20 C to 70 C Viral nucleic acid eluate too dilute Use recommended 30 50u of Eluent Improper elution conditions Apply preheated Eluent into the center of Spin Column membrane as described on page 5 Column Starting material was not completely disrupted clogging Reduce the amount of starting material and increase disruption time Degraded RNA Precipitates were not removed When using plasma samples remove visible kryoprecipitates by centrifugation for 5 min at 3000 x g RNase contamination To avoid RNase contamination wear gloves during all procedure and change gloves frequently
10. ld be stored at room temperature until itis used for sample processing Load the sample to an empty 2 0 ml lysis tube Add the same volume 1 1 of Lysis Solution supplemented with Carrier RNA and 50 ul of Proteinase K mix thoroughly by vortexing or pipetting Incubate the sample for 15 min at 56 C in a thermomixer Leave thermomixer turned on for Eluent preheating during later steps of the procedure Centrifuge for 3 5 s at full speed to collect any sample solution from the inside of the lid Note e Supplement Lysis Solution with Carrier RNA prior to use page 6 Use the appropriate internal control as required in a downstream assay user s manual Do not add the internal control directly to plasma samples Do not add Proteinase K directly to Lysis Solution Add ethanol 96 100 Use 150 pl ethanol for every 100 yl of initial sample volume Mix by pipetting or vortexing Incubate the sample at room temperature for 3 min Centrifuge for 3 5 s at full speed to collect drops from the inside of the lid Transfer the lysate to the prepared Spin Column preassembled within the wash tube Do not load more than 700 ul of lysate on the column For larger volumes load the remaining volume of the lysate to the same column and centrifuge for a second time Centrifuge the column for 1 min at 6 000 x g Discard the flow through Place the Spin Column into a new 2 ml Wash Tube Go to step 5 of the standard Viral Nucleic Acid Purificat
11. rification from urine Step 1 Procedure Add 0 5 ml of 0 5 M EDTA to 4 5 ml of urine final concentration 50 mM Note Urine samples may contain insoluble salt precipitates that can reduce nucleic acid yields thus limiting sample volume used for purification Centrifuge 10 min at 800 x g 3 000 rpm Discard the supernatant 01 amp G N Resuspend the pellet in 200 wl of 1x PBS Go to step 1 ofthe standard Viral Nucleic Acid Purification P rotocol page 7 Note Urine samples larger than 4 5 ml are not recommended D Nucleic acid purification from saliva Step Procedure 1 To collect cells centrifuge the saliva sample for 5 min at 3 000 x g 2 Resuspend cells in 200 ul of 1 x PBS or TE buffer 3 Gotostep 1 of the standard Viral Nucleic Acid Purification Protocol page 7 E Viral nucleic acid purification protocol for larger sample volumes up to 400 pl Important Lysis Solution and ethanol are the only components that have to be scaled up Step Procedure 1 Add 50 ul of Column Preparation Liquid to the center of Spin Column membrane so that the membrane is entirely moistened Note e Before starting the procedure each new Spin Column must be prepared by treating it with Column Preparation Liquid Column treatment maximizes binding of the nucleic acids to the membrane resulting in more consistent yields e Do not centrifuge the prepared column The prepared column shou
12. s hazardous waste 12 Proteinase K Xn Harmful Hazard determining component of labeling Proteinase Tritirachium album serine Risk phrases R42 May cause sensitization by inhalation Safety phrases 23 Do not breathe gas fumes vapor spray 36 Wear suitable protective clothing 45 In case of accident or if you feel unwell seek medical advice immediately show the label where possible 60 This material and its container must be disposed of as hazardous waste REFERENCES 1 Boom R C J A Sol M M M Salimans C L J ansen P M E W Dillen and J van der Noordaa 1990 Rapid and simple method for purification of nucleic acids J Clin Microbiol 28 495 503 PRODUCT USE LIMITATION This product is developed designed and sold exclusively for research purposes and in vitro use only The product was not tested for use in diagnostics or for drug development nor is it suitable for administration to humans or animals Please refer to www thermoscientific com onebio for Material Safety Data Sheet of the product 2012 Thermo Fisher Scientific Inc All rights reserved All trademarks are the property of Thermo Fisher Scientific Inc and its subsidiaries 13
13. sed Y calculated volume ml of Lysis Solution Z volume ul of Carrier RNA to add to Y ml of Lysis Solution INTERNAL CONTROL The presence of an internal control throughout the extraction and purification procedure may be necessary for certain assays Please refer to the user manual provided with the downstream detection assay for further directions on how to use an internal control ADDITIONAL MATERIALS AND EQUIPMENT REQUIRED Pipettes and sterile nuclease free pipette tips with aerosol barrier Vortex Ethanol 96 100 Microcentrifuge Thermomixer Disposable gloves Measuring cylinder Nuclease free microcentrifuge tubes of an appropriate size for preparing mixtures of Carrier RNA and Lysis Solution Microcentrifuge tubes for sample lysis and elution e RNase free tubes should be used for RNA elution PROTOCOLS Before starting e Readthe user manual make sure all the directions are followed as indicated e Make sure all working solutions and samples have been prepared according to recommendations page 5 6 e Ensure all necessary equipment and additional materials are available before beginning the procedure page 6 e Organize proper handling of potentially infectious waste before beginning the procedure e Follow recommended safety instructions as you will be working with potentially infectious material e Attention and care must be taken during the entire process e All centrifugation steps must be performed at room
14. temperature A Viral nucleic acid purification main protocol This protocol is for viral DNA and RNA purification from 200 ul of EDTA or citrate treated plasma serum blood or milk samples For other sample types refer to additional protocols page 9 Following procedure provides instruction for processing one sample When using larger than 200 ul up to 400 ul sample volumes refer to protocol E page 10 Step Procedure Add 50 ul of Column P reparation Liquid to the center of Spin Column membrane so that the membrane is entirely moistened Note e Before starting the procedure each new Spin Column must be prepared by treating it 1 with Column Preparation Liquid Column treatment maximizes binding of the nucleic acids to the membrane resulting in more consistent yields e Do not centrifuge the prepared column The prepared column should be stored at room temperature until itis used for sample processing Load 200 ul of sample to an empty 1 5 ml lysis tube Add 200 ul of Lysis Solution supplemented with Carrier RNA and 50 ul of Proteinase K mix thoroughly by vortexing or pipetting Incubate the sample for 15 min at 56 C in a thermomixer Leave thermomixer turned on for Eluent preheating during later steps of the procedure 2 Centrifuge for 3 5 s at full speed to collect any sample solution from the inside of the lid Note e Supplement Lysis Solution with Carrier RNA prior to use page 6 Use the appropriate internal con
15. trol as required in a downstream assay user s manual Do not add the internal control directly to plasma samples Do not add Proteinase K directly to Lysis Solution Add 300 ul of ethanol 96 100 and mix by pipetting or vortexing 3 Incubate the sample at room temperature for 3 min Centrifuge for 3 5 s at full speed to collect drops from the inside of the lid Transfer the lysate to the prepared Spin Column preassembled within the wash tube 4 Centrifuge the column for 1 min at 6 000 x g Discard the Wash Tube containing flow through Place the Spin Column into a new 2 ml Wash Tube 7 Note e Ensure that a new Spin Column has been prepared as described in step 1 Add 700 ul of Wash Buffer 1 supplemented with ethanol to the S pin Column Centrifuge the column for 1 min at 6 000 x g Discard the Wash Tube containing flow through Place the Spin Column into a new 2 ml Wash Tube Note e Supplement concentrated Wash Buffer 1 with ethanol prior to the first use P age 5 Add 500 ul of Wash Buffer 2 supplemented with ethanol to the S pin Column Centrifuge the column for 1 min at 6 000 x g Discard the Wash Tube containing flow through Place the Spin Column into a new 2 ml Wash Tube Note e Supplement concentrated Wash Buffer 2 with ethanol prior to the first use P age 5 Add 500 ul of Wash Buffer 2 supplemented with ethanol to the S pin Column Centrifuge the column for 1 min at 6 000 x g Discard the Wash Tu
16. uffer 1 Wash Buffer 2 Concentrated wash buffer 25 ml 11 ml Ethanol 96 100 15 ml 44 ml Total volume 40 ml 55 ml e Ensure all working solutions are prepared according to the recommendations in the protocol e After preparing each solution mark the bottle to indicate that this step has been completed e Check all solutions in the kit for any salt precipitation before each use Re dissolve any precipitate by warming the solution to 37 C and then equilibrate to room temperature 20 5 C e For swabs or collected cells reconstitute cells to recommended sample volume using PBS 137 mM NaCl 2 7 mM KCI 10 mM NazHP Oa 2 mM KHP O4 pH 7 4 e tis the user s responsibility to use appropriate controls during the procedure CARRIER RNA Usage of Carrier RNA is important for efficient recovery of viral nucleic acids for two reasons First Carrier RNA facilitates binding of viral nucleic acids to the silica membrane especially when there are only a small number of viral nucleic acid molecules in the sample Additionally in the rare event when there are a small number of active RNase molecules large amounts of Carrier RNA reduce the probability of viral RNA being degraded under chaotropic conditions If Carrier RNA is not added to the Lysis Solution reduced viral nucleic acid yields may result PREPARATION OF CARRIER RNA Carrier RNA is provided in a dried state packed in a moisture impermeable aluminum bag Prior to the first use
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