English handbook article No. D-2021
Contents
1. V120222 Page 3 17 Functional principle The chemagic Prepito NA Body Fluid D200 Kit is based on chemagen s proprietary magnetic bead technology platform Cells or viruses in the sample material are lysed during the isolation process The released nucleic acids bind to small magnetisable particles which are then magnetically separated from the sample material During subsequent steps contaminations are removed and the purified nucleic acids are transferred into an elution medium The automated sample processing by the chemagic Prepito D excludes cross contamination and ensures a safe handling of infectious sample material Quality control Each lot is tested for its defined specifications according to chemagen s Quality Management System Procedures that are not in accordance with this manual could cause inadequate results Product limitations Choice of protocol The Kit is designed for the use with different kinds of body fluids such as whole blood fresh or frozen plasma serum urine liquor but also for different kinds of swabs and feces suspensions Depending on the sample material different protocols have to be used e Protocol Body Fluid Blood for whole blood and sample material contaminated with whole blood In the following protocol description these samples are named with the term Blood Samples e Protocol Body Fluid Plasma for other sample material as described above In the following protocol description these sampl2. Tubes contain air after connecting the chemagic 8 Pack to the machine Fill the tubes completely using the manual priming function Buffers in the chemagic 8 Pack are empty Change the chemagic 8 Pack Dont use the chemagic 8 Pack for more than the indicated preparations Buffers of the chemagic 8 Pack are not connected in the right manner to the chemagic Prepito D Check correct the connections between the chemagic Prepito D and the chemagic 8 Pack Irregular dispensing of the buffers Check the calibration of the pumps Blood samples are not mixed thoroughly Mix the blood samples thoroughly Quotient of absorption Azgo Azgo to low Traces of Magnetic Beads in the elution solution falsify the result of the measurement Remove remaining traces of Magnetic Beads using a manual separator e g chemagic Stand 2x12 art No 300 Contaminated or inactive Proteinase K Visible microbial growth in Proteinase K solution Use sterile water for resuspension of the Proteinase K V120222 Page 16 17 Problem Possible Cause Recommendation Solution Contaminated or inactive Proteinase K Incorrect storage of the Proteinase K solutions Store Proteinase K solution at 4 do not use the solutions longer than 6 weeks Store aliquots at 20 Avoid thawing freezing cycles Subsequent detection reactions are not working optimally Insufficient amount of nucleic acids
3. amount of Magnetic Beads added Resuspend the Magnetic Beads well before adding to the lysate Insufficient lysis Add the correct volume of lysis buffer Buffers in the chemagic 8 Pack are not connected to the machine Connect the buffers in the chemagic 8 Pack to the machine The chemagic 8 Pack is not positioned in the right manner on the reagent holder Place the chemagic 8 Pack in the correct position on the reagent holder Tubes contain air after connecting the chemagic 8 Pack to the machine Fill the tubes completely using the manual priming function Buffers in the chemagic 8 Pack are empty Change the chemagic 8 Pack Don t use the chemagic 8 Pack for more than the indicated preparations Buffers of the chemagic 8 Pack are not connected in the right manner to the chemagic Prepito D Check correct the connections between the chemagic Prepito D and the chemagic 8 Pack Irregular dispensing of the buffers Check the calibration of the pumps Insufficient yields of DNA blood samples Incorrect amount of Magnetic Beads added Resuspend the Magnetic Beads well before adding to the lysate Buffers in the chemagic 8 Pack are not connected to the machine Connect the buffers in the chemagic 8 Pack to the machine The chemagic 8 Pack is not positioned in the right manner on the reagent holder Place the chemagic 8 Pack in the correct position on the reagent holder
4. eee treat a tees 2 intended USE ca iaia iaia aldeidi ani 2 Functional principles asas isso aaa liane 4 Quality contol saias ella a a GO LAS iota e nad 4 Product limitations Choice of protocol err rareaaaaraananeaaarea near nanaaaaa anna 4 Stability and SO e ar a acter a eae Peder Sheet oi alae 4 Protocol CUPatlon erine E EA E T lar PR 5 Typical yields whole blood SAMPLES 0 0 0 eee eeeeteeeeeeeeeteeeeeeeeeeeeeaeeeeeeaaeeeseeaaeeeeeeeaaeeeseeaaeeeseeaaeeesneaeeeseaas 5 Contents of the Kit unit corresp to 180 preparations from 200 uL sample material 6 Safety a ilo sfintere ata a a Sl e le ini lele ivi iaia 8 Equipment and other material to be provided by the user i 10 Purification Protocol using the chemagic Prepito D i 10 Positioning of the Deep Well Plate and the chemagic Tip amp Tube Rack 10 Protocol Steps Blood Samples chemagic Prepito D serial numbers 1 99 11 Protocol Steps Plasma Samples chemagic Prepito D serial numbers 1 99 ceeeeeeeeeseeeeees 12 Protocol Steps Blood Samples chemagic Prepito D serial numbers 100 and later 13 Protocol Steps Plasma Samples chemagic Prepito D serial numbers 100 and later 14 ECCI RINTE e EE A ina eli ili iii i Li i e ei e i iii bu 15 UV Measurements 0 avatar Ania iaia a Sia 15 Troubleshootinig iuisioio Alia aa ia 16
5. Caps 180 included in the chemagic 8 Pack V120222 Page 7 17 Safety To avoid injuries while working with the kit components always wear safety glasses disposable gloves and protective clothing For detailed information please refer to the according material safety data sheet MSDS Reagent 1 Magnetic Beads no hazardous ingredients Reagent 2 Lysis Buffer 1 blood Guanidine hydrochloride CAS No 50 01 1 EC No 200 002 3 Xn R22 36 38 S2 22 Triton X 100 CAS No 9002 93 1 EC No R22 41 S24 26 39 Reagent 3 Lysis Buffer 1 plasma Guanidine thiocyanate CAS No 593 84 0 EC No 209 812 1 Xn R20 21 22 32 52 53 S13 61 Reagent 4 Binding Buffer 2 blood Sodium Perchlorate CAS No 7601 89 0 EC No 231 511 9 Xn R9 22 S13 22 27 Ethanol CAS No 64 17 5 EC No 200 578 6 F R10 S7 16 Reagent 5 Binding Buffer 2 plasma Sodium Perchlorate CAS No 7601 89 0 EC No 231 511 9 Xn R9 22 S13 22 27 Ethanol CAS No 64 17 5 EC No 200 578 6 F R10 Reagent 6 Wash Buffer 3 Ethanol CAS No 64 17 5 EC No 200 578 6 F R10 S7 16 Guanidin Hydrochlorid CAS No 50 01 1 EC No 200 002 3 Xn R22 36 38 S2 22 Sodium Perchlorate CAS No 7601 89 0 EC No 231 511 9 Xn R9 22 S13 22 27 Reagent 7 Wash Buffer 4 Ethanol CAS No 64 17 5 EC No 200 578 6 F R10 S7 16 Sodium Perchlorate CAS No 7601 89 0 EC No 231 511 9 Xn R9 22 S13 22 27 Reagent 8 Wash Buffer 5 Ethanol CAS No 64 17 5 EC No 200 578 6 F R10 S7 16 Reagent 9 Wash Buffer 6 no
6. Prepito NA Body Fluid D200 Kit art No D 2021 CE P PerkinElmer Symbols V em Kit contains reagents for 180 preparations Ch Refer to information given in the handbook V120222 Expiry date LOT Lot number IVD in vitro diagnostic medical device X Temperature limitations REF D 2021 all PerkinElmer chemagen Technologie GmbH Arnold Sommerfeld Ring 2 D 52499 Baesweiler Tel 49 2401 805500 Intended use With the Prepito NA Body Fluid D200 Kit nucleic acids can be isolated from different kinds of body fluids such as whole blood plasma serum urine liquor but also for different kind of swabs and feces suspensions for subsequent in vitro diagnostic purposes The Kit has to be used with the chemagic Prepito D The product is intended for professional users such as technicians and physicians trained in molecular biology techniques To minimize irregularities in diagnostic results the product should always be used with an internal control as well as positive and negative controls throughout the process of sample preparation sample amplification and detection according to the downstream assay used Any diagnostic results generated using the sample preparation procedure in conjunction with any downstream diagnostic NAT assay should be interpreted with regard to other clinical or laboratory findings V120222 Page 2 17 Content SymbOIS 22 4 st ctet cs lie li Aa era ce Sad eg aaa E Gea Lelio amelia
7. Tip amp Tube Rack on its default position on the tracking system Check the accurate fit of the DWP and chemagic Tip amp Tube Rack and lock both by closing the safety latch Close the front door and immediately start the automated isolation process by pressing start V120222 Page 11 17 Protocol Steps Plasma Samples chemagic Prepito D serial numbers 1 99 1 2 3 4 5 6 10 11 12 18 14 15 Switch on the chemagic Prepito D and wait for the self test to finish Press change protocol Select the Prepito NA Body Fluid D200 Kit protocol by pressing Body Fluid Plasma Enter the access code 3005 for authorization and confirm by pressing enter Confirm the selection of the correct protocol by pressing enter Read the protocol information in the appearing information screen Confirm by pressing continue Select the sample positions and confirm by pressing continue Enter the kit barcode with the barcode scanner and confirm by pressing ok For the registration of the samples and storage tubes press yes and follow the instructions on the touch screen panel to enter the according barcodes Prepare the chemagic Tip amp Tube Rack with the required materials Place one 0 75 mL reaction tube filled with 50 100 uL Elution Buffer position 1 one 0 75 mL reaction tube filled with 150 uL of Magnetic Beads solution position 2 and one Disposable Tip position 3 for each sample into positio
8. ep Well Plate and chemagic Tip amp Tube Rack Use Poly A RNA and Protease solutions only with samples contaminated with blood Don t use Proteinase K and Poly A RNA for the preparation of whole blood material Add 200 uL sample material into the sample position of the Deep Well Plate see section above Positioning of Deep Well Plate and chemagic Tip amp Tube Rack An information screen indicates the previously selected sample positions Ensure that the sample positions in the DWP correspond to the selected positions Place the DWP on its default position on the tracking system and press Continue Place the chemagic Tip amp Tube Rack on its default position on the tracking system Check for accurate fit of the DWP and chemagic Tip amp Tube Rack and lock both by closing the safety latch Close the front door and start the automated isolation process by pressing Start immediately V120222 Page 13 17 Protocol Steps Plasma Samples chemagic Prepito D serial numbers 100 and later 1 P So nm oN oo a 10 11 12 18 14 15 16 17 Switch on the chemagic Prepito D and wait for the self test to finish Press Change Protocol Press Body Fluid in the Select Protocol Group window Select the Prepito NA Body Fluid D200 Kit protocol by pressing BF Plasma and confirm by pressing OK Confirm the protocol selection in the Select Protocol Group window by pressing OK Enter the 4 digi
9. es are named with the term Plasma Samples Body fluids can directly be used in aliquots of 200 uL per isolation Transport media from swab samples can either be processed directly or the cells can be concentrated by a centrifugation step In either case the processable volume per sample is 200 uL Feces suspensions have to be centrifuged and 200 uL of the supernatant have to be used per isolation The Kit is not intended for the use with tissue as sample material The isolation efficiency with other types of sample material has not been determined In some rare cases especially with compromised blood aged or improperly stored colored eluates can be observed Colored eluates may interfere with UV measurements and may affect results in subsequent downstream applications Stability and storage Expiry dates are stated on the box and the single components of the kit Do not use any components of the kit beyond the expiration date All kit components can be stored at room temperature Lysis Buffer 1 plasma and Poly A RNA Buffer have to be stored in the dark Lysis Buffer 1 may form a precipitate upon storage If necessary warm to approximately 55 to redissolve Precipitates in the Poly A RNA buffer can be redissolved at room temperature After reconstitution Proteinase K solution and Poly A RNA solution have to be stored at 4 The solutions can be used for 6 weeks For long term storage we recommend aliquoting the Proteinase K s
10. hazardous ingredients Reagent 10 Elution Buffer 7 no hazardous ingredients V120222 Page 8 17 Reagent 10 Proteinase K Protease CAS No 39450 01 6 EC No 254 457 8 Xn R36 37 38 42 43 S22 24 26 36 37 39 46 EC 1272 2008 H315 H317 H319 H334 H335 P261 P280 P285 P305 P351 P338 P321 P405 Reagent 12 Poly A RNA no hazardous ingredients Reagent 13 Poly A RNA Buffer Guanidine Thiocyanate CAS No 593 84 0 EC No 209 812 1 Xn R20 21 22 32 52 53 S13 61 V120222 Page 9 17 Equipment and other material to be provided by the user chemagic Prepito D RNAse free water disposable gloves pipette and pipette tips with aerosol barrier ensure that all used material is RNase free Purification Protocol using the chemagic Prepito D The protocol is suitable for up to 12 samples in parallel see protocol steps below Detailed instructions for the use of the chemagic Prepito D are given in the corresponding user manual Before you start e Check all kit components for integrity In case of damages contact your supplier e Connect the tubes according to their numbering to the respective counterparts at the chemagic 8 Pack Remove the lids from the individual buffer bottles in the chemagic 8 Pack and pierce the septum with the spike at the end of the tube Place the chemagic 8 Pack upside down on the reagent holder e Dissolve the lyophilized Proteinase K in RNAse free water see instruction on the tube and Poly A RNA in 440 uL Pol
11. ns according to the sample positions Shake the Magnetic Bead solution vigorously until all Magnetic Beads are completely suspended An incomplete resuspension of the Magnetic Bead solution could cause a decreased yield of extracted nucleic acids Add 10 uL of Proteinase K and 4 uL Poly A RNA solutions to each well of the Deep Well Plate DWP defined as sample well see above section Positioning of the Deep Well Plate and the chemagic Tip amp Tube Rack Add 450 uL Lysis Buffer plasma and 200 uL sample material to each sample well prefilled with Proteinase K and Poly A RNA solutions An information screen indicates the previously selected sample positions Ensure that the sample positions in the DWP correspond to the selected positions Place the DWP on its default position on the tracking system and press continue Place the chemagic Tip amp Tube Rack on its default position on the tracking system Check the accurate fit of the DWP and chemagic Tip amp Tube Rack and lock both by closing the safety latch Close the front door and immediately start the automated isolation process by pressing start V120222 Page 12 17 Protocol Steps Blood Samples chemagic Prepito D serial numbers 100 and later 1 P SO N oN o a 10 11 12 18 14 15 16 17 Switch on the chemagic Prepito D and wait for the self test to finish Press Change Protocol Press Body Fluid in the Select Protocol Group
12. olution and the Poly A RNA solution and storing at 20 V120222 Page 4 17 Protocol duration The length of the purification protocol is 75 min Typical yields whole blood samples From 200 uL whole blood 4 8 ug DNA can be isolated The obtained yields depend on the number of leukocytes in the sample material and vary from specimen to specimen V120222 Page 5 17 Contents of the Kit unit corresp to 180 preparations from 200 uL sample material 1 2 10 11 12 13 14 V120222 Magnetic Beads 30 mL Lysis Buffer 1 blood 95 mL Guanidine hydrochloride 15 30 Triton X 100 1 3 Lysis Buffer 1 plasma 2 x 48 mL Guanidine thiocyanate 43 50 Binding Buffer 2 blood 210 mL Tris HCl buffer Sodium perchlorate 30 40 Ethanol 35 45 Binding Buffer 2 plasma 210 mL Tris HCl buffer Sodium perchlorate 25 28 Ethanol 45 60 Wash Buffer 3 210 mL Tris HCl buffer Guanidine hydrochloride 3 5 Sodium perchlorate 15 20 Ethanol 20 25 Wash Buffer 4 110 mL Tris HCl buffer Sodium perchlorate 10 15 Ethanol 20 25 Wash Buffer 5 110 mL Ethanol 70 80 Wash Buffer 6 110 mL Elution Buffer 7 50 mL 10 mM Tris HCl buffer pH 8 0 Proteinase K 2 0 mL Poly A RNA 2 x 350 ug Poly A RNA Buffer 2 x 440 uL Disposable Tips 180 Page 6 17 15 2 mL Deep Well Plates 15 16 0 75 mL Reaction Tubes 360 17 0 75 mL
13. on the touch screen panel to enter the according barcodes Prepare the chemagic Tip amp Tube Rack with the required materials Place one 0 75 mL reaction tube filled with 50 100 uL Elution Buffer position 1 one 0 75 mL reaction tube filled with 150 uL of Magnetic Beads position 2 and one Disposable Tip position 3 for each sample into positions according to the sample positions Shake the Magnetic Bead solution vigorously until all Magnetic Beads are completely suspended An incomplete resuspension of the Magnetic Bead solution could cause a decreased yield of extracted nucleic acids For the processing of sample material contaminated with blood add 4 uL Poly A RNA solution and 10 uL Proteinase K solution into the sample position of the Deep Well Plate DWP see section above Positioning of Deep Well Plate and chemagic Tip amp Tube Rack Use Poly A RNA and Proteinase K solutions only with samples contaminated with blood Don t use Proteinase K and Poly A RNA for the preparation of whole blood material Add 200 uL sample material into the sample position of the Deep Well Plate see section above Positioning of Deep Well Plate and chemagic Tip amp Tube Rack An information screen indicates the previously selected sample positions Ensure that the sample positions in the DWP correspond to the selected positions Place the DWP on its default position on the tracking system and press continue Place the chemagic
14. ositions Ensure that the sample positions in the DWP correspond to the selected positions Place the DWP on its default position on the tracking system and press Continue Place the chemagic Tip amp Tube Rack on its default position on the tracking system Check for accurate fit of the DWP and chemagic Tip amp Tube Rack and lock both by closing the safety latch Close the front door and start the automated isolation process by pressing Start immediately V120222 Page 14 17 General remarks It is strongly recommended to use the extracted nucleic acids immediately for amplification If nucleic acid extracts cannot be used for amplification directly after preparation the nucleic acid extracts can be kept at 20 or preferably at 70 for up to one month or one year respectively The Elution Buffer included in this kit is 10 mM Tris HCl pH 8 0 UV Measurements In some cases you may find traces of magnetic beads left in the eluate Such particles will not interfere with PCR and most downstream applications but may increase the background in UV measurements In such a case prior to UV analysis we recommend an additional separation step using a manual separator e g chemagic Stand 2x12 art No 300 in order to separate any traces of particles V120222 Page 15 17 Troubleshooting Problem Possible Cause Recommendation Solution Low detection sensitivity for positive controls and or target nucleic acid Incorrect
15. t access code 3005 for authorization and confirm by pressing Enter Press Start Process Read the protocol information in the appearing information screen and confirm by pressing Continue Select the sample positions and confirm by pressing OK Enter the kit barcode with the barcode scanner and confirm by pressing OK For the registration of the samples and the storage tubes press Yes and follow the instructions on the touch screen panel to enter the according barcodes Prepare the chemagic Tip amp Tube Rack with the required materials Place one 0 75 mL reaction tube filled with 50 100 uL Elution Buffer position 1 one 0 75 mL reaction tube filled with 150 uL of Magnetic Beads solution position 2 and one Disposable Tip position 3 for each sample into positions according to the sample positions Shake the Magnetic Bead solution vigorously until all Magnetic Beads are completely suspended An incomplete resuspension of the Magnetic Bead solution could cause a decreased yield of extracted nucleic acids Add 10 uL of Proteinase K and 4 uL Poly A RNA solutions to each well of the Deep Well Plate DWP defined as sample well see above section Positioning of the Deep Well Plate and the chemagic Tip amp Tube Rack Add 450 uL Lysis Buffer plasma and 200 uL sample material to each sample well prefilled with Proteinase K and Poly A RNA solutions An information screen indicates the previously selected sample p
16. used for the detection reaction Too much nucleic acids used for the detection reaction Determine the concentration of nucleic acids via UV measurement and use an adequate amount in subsequent reactions Red eluates from plasma or serum samples low detection sensitivity Traces of erythrocytes in the plasma or serum samples Avoid to carry over erythrocytes during the preparation of plasma or serum Brown or red eluates from whole blood samples In some rare cases especially with compromised blood aged or improperly stored colored eluates can be observed Don t use improperly stored blood Don t use Proteinase K and Poly A RNA for the preparation of whole blood material Blood was prepared with Lysis Buffer 1 plasma and the Body Fluid Plasma protocol Ensure that blood samples are prepared with the Body Fluid Blood protocol Malfunction of the instrument e g mechanical electrical or electronical problems Contact chemagen or your local supplier detailed information is given in the manual of the chemagic Prepito D V120222 Page 17 17
17. window Select the Prepito NA Body Fluid D200 Kit protocol by pressing BF Blood and confirm by pressing OK Confirm the protocol selection in the Select Protocol Group window by pressing OK Enter the 4 digit access code 3005 for authorization and confirm by pressing Enter Press Start Process Read the protocol information in the appearing information screen and confirm by pressing Continue Select the sample positions and confirm by pressing OK Enter the kit barcode with the barcode scanner and confirm by pressing OK For the registration of the samples and the storage tubes press Yes and follow the instructions on the touch screen panel to enter the according barcodes Prepare the chemagic Tip amp Tube Rack with the required materials Place one 0 75 mL reaction tube filled with 50 100 uL Elution Buffer position 1 one 0 75 mL reaction tube filled with 150 uL of Magnetic Beads position 2 and one Disposable Tip position 3 for each sample into positions according to the sample positions Shake the Magnetic Bead solution vigorously until all Magnetic Beads are completely suspended An incomplete resuspension of the Magnetic Bead solution could cause a decreased yield of extracted nucleic acids For the processing of sample material contaminated with blood add 4 uL Poly A RNA solution and 10 uL Proteinase K solution into the sample position of the Deep Well Plate DWP see section above Positioning of De
18. y A RNA Buffer per tube Positioning of the Deep Well Plate and the chemagic Tip amp Tube Rack The following scheme shows the orientation of the 96 Deep Well Plate DWP and the chemagic Tip amp Tube Rack For detailed information see protocols steps jo amp A S Q Vv O 1 Co O 200 uL sample material optional Proteinase K Poly A RNA 2 Pos 4 second row for Disposable Tips not used in this protocol gt Pos Disposable Tips E x os 3 p p o Pos 2 0 75 mL reaction tubes with 150 uL Magnetic Beads q Pos 1 0 75 mL reaction tubes with 50 100 uL Elution Buffer V120222 Page 10 17 Protocol Steps Blood Samples chemagic Prepito D serial numbers 1 99 1 2 3 4 5 6 10 11 12 18 14 15 Switch on the chemagic Prepito D and wait for the self test to finish Press change protocol Select the Prepito NA Body Fluid D200 Kit protocol by pressing Body Fluid Blood Enter the access code 3005 for authorization and confirm by pressing enter Confirm the selection of the correct protocol by pressing enter Read the protocol information in the appearing information screen Confirm by pressing continue Select the sample positions and confirm by pressing continue Enter the kit barcode with the barcode scanner and confirm by pressing ok For the registration of the samples and storage tubes press yes and follow the instructions
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