MagNA Pure Compact Nucleic Acid Isolation Kit I -...

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For general laboratory use. R

MagNA Pure Compact Nucleic Acid Isolation Kit I

y Version: 16Content version: November 2016

Prefilled reagents for the isolation of genomic DNA from mammalian whole blood or cultured cells, total nucleic acids from mammalian serum or plasma, or bacterial DNA (1) (from different types of sample of mammalian origin or bacterial cultures) using the MagNA Pure Compact Instrument

Only in combination with the MagNA Pure Bacteria Lysis Buffer (Cat. No. 04 659 180 001)

Cat. No. 03 730 964 001 1 kit 32 isolations 4 x 8 sealed cartridges

Store the kit at +15 to +25°C.

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y Version: 16 MagNA Pure Compact Nucleic Acid Isolation Kit I

1. General Information ............................................................................................................................31.1. Contents ................................................................................................................................................................................................... 31.2. Storage and Stability ........................................................................................................................................................................... 3

Storage Conditions (Product) .......................................................................................................................................................... 31.3. Additional Equipment and Reagents Required ......................................................................................................................... 31.4. Application .............................................................................................................................................................................................. 41.5. Preparation Time ................................................................................................................................................................................... 4

Assay Time .............................................................................................................................................................................................. 4

2. How to Use this Product ....................................................................................................................52.1. Before you Begin .................................................................................................................................................................................. 5

Sample Materials .................................................................................................................................................................................. 5Control Reactions ................................................................................................................................................................................. 5Internal Control ...................................................................................................................................................................................... 6Two Possible Methods for Incorporating the IC: ....................................................................................................................... 6General Considerations ...................................................................................................................................................................... 6Precautions ............................................................................................................................................................................................. 7Prevention of Cross-Contamination ............................................................................................................................................... 7Safety Information ................................................................................................................................................................................ 7Laboratory Procedures ....................................................................................................................................................................... 7Waste Handling ..................................................................................................................................................................................... 7

2.2. Protocols .................................................................................................................................................................................................. 8External Lysis Protocol ........................................................................................................................................................................ 8Blood Cells .............................................................................................................................................................................................. 8Purification Protocols from Bacterial-Containing Sources ................................................................................................... 8Overview of Treatments For Bacterial Sample Materials With MagNA Pure Bacteria Lysis Buffer ..................... 9MagNA Pure Compact Protocol Using the MagNA Pure Compact Nucleic Acid Isolation Kit I ..........................12

3. Results ................................................................................................................................................ 15DNA Yield and Purity ........................................................................................................................................................................15DNA Integrity .......................................................................................................................................................................................15Sensitivity ...............................................................................................................................................................................................15Scalability ...............................................................................................................................................................................................16

4. Troubleshooting ................................................................................................................................ 17

5. Additional Information on this Product ....................................................................................... 195.1. Test Principle ........................................................................................................................................................................................19

How this Product Works ...................................................................................................................................................................195.2. Quality Control .....................................................................................................................................................................................19

6. Supplementary Information ........................................................................................................... 206.1. Conventions ..........................................................................................................................................................................................206.2. Changes to previous version ..........................................................................................................................................................206.3. Ordering Information .........................................................................................................................................................................206.4. Trademarks ............................................................................................................................................................................................216.5. License Disclaimer .............................................................................................................................................................................216.6. Regulatory Disclaimer .......................................................................................................................................................................216.7. Safety Data Sheet ...............................................................................................................................................................................216.8. Contact and Support .........................................................................................................................................................................21


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1. General Information

y Version: 16MagNA Pure Compact Nucleic Acid Isolation Kit I


1.1. Contents

Label Function ContentReagent Cartridge Contains reagents sufficient for one isolation run

(well 1: Proteinase K; well 2: Lysis Buffer; well 3: MGPs; wells 4,5: Wash Buffer I; well 6: Wash Buffer II; well 7: Wash Buffer III; well 8: Elution Buffer).

32 sealed cartridges

Tip Tray Contains Reaction Tips (2 large and 1 small) and Piercing Tool. 32 disposable Tip TraysMagNA Pure Tube 2.0mL To be placed into the Tube Rack of the MagNA Pure Compact

Instrument (see Operator’s Manual). To be placed into the Elution Tube Rack of the MagNA Pure Compact Instrument (see Operator’s Manual).

2 × 35 barcoded tubes, 2.0 ml

MagNA Pure Tube Cap To seal the MagNA Pure Tube 2.0mL containing the eluates. 35 tube caps

Fig. 1: Reagent Cartridge of the MagNA Pure Compact Nucleic Acid Isolation Kit I

1.2. Storage and Stability

Storage Conditions (Product)The kit is shipped at ambient temperature.

Unopened components of the MagNA Pure Compact Nucleic Acid Isolation Kit I are stable at +15 to +25°C until the expiration date printed on the label.

1.3. Additional Equipment and Reagents RequiredAdditional reagents and equipment required to perform nucleic acid isolations with the MagNA Pure Compact Nucleic Acid Isolation Kit I using the MagNA Pure Compact Instrument:

Standard Laboratory Equipment

• Pipettes and nuclease-free, aerosol-preventive tips to predispense samples into the Sample Tubes• Centrifuge and suitable tubes• Optional when using an Internal Control: 2.0 ml Sarstedt Tubes (without cap: Sarstedt #72.608; with cap: Sarstedt

#72.693)• Optional when using a purification protocol with external lysis: MagNA Pure LC DNA Isolation Kit I – Lysis/Binding

Buffer Refill*





For Isolation of DNA from Blood Cells (Optional)

• PBS*• Red Blood Cell Lysis Buffer* (for pre-isolation of WBCs)• Roller incubator (for pre-isolation of WBCs)• Vacutainer CPT tube (BD Diagnostics; for pre-isolation of PBMCs)• Hemocytometer (e.g., Neubauer device with counting chambers)

For Isolation of Bacterial DNA (Optional)

• MagNA Pure Bacteria Lysis Buffer*• MagNA Lyser Instrument* with MagNA Lyser Green Beads*• Proteinase K*• Heat block for 1.5 ml (or 2 ml) reaction tubes

1.4. ApplicationThe MagNA Pure Compact Nucleic Acid Isolation Kit I, a General Purpose Reagent (GPR), is specifically designed to isolate highly purified genomic DNA from mammalian whole blood, blood cells, buffy coat or cultured cells, and total nucleic acids (e.g., viral DNA and RNA) from mammalian serum or plasma, using the MagNA Pure Compact Instrument. In combination with the MagNA Pure Bacteria Lysis Buffer* and the DNA_Bacteria purification protocol, bacterial DNA can be isolated from different types of sample of mammalian origin (such as urine, BAL, sputum, CSF, swabs) or bacterial cultures. The purified nucleic acids can be used in PCR or RT-PCR on the LightCycler® Instruments, standard thermal block cyclers, or in other typical downstream applications. Purified nucleic acid is free of any PCR inhibitors.

The MagNA Pure Compact Nucleic Acid Isolation Kit I cannot be used for co-isolation of RNA from mammalian whole blood or cultured cells.

Isolation reagents are provided in prefilled, sealed, and barcoded reagent cartridges. Each cartridge contains all reagents required for a single isolation. Reaction tips are provided in a disposable tip tray. Sterile tubes for samples and purified nucleic acid eluates are barcoded. The kit is designed for 32 isolations (4 × 8) from:• 100 – 400 μl mammalian whole blood (containing no more than 5 × 103 cells/μl)• 100 – 400 μl suspension of mammalian blood cells (white blood cells [WBCs] or peripheral blood mononuclear cells

[PBMCs] or buffy coat [containing no more than 5 × 103 cells/μl])• 100 – 400 μl mammalian serum or plasma• 100 μl suspension of cultured cells (containing no more than 1 × 106 cells)

For isolation of bacterial DNA, in combination with the MagNA Pure Bacteria Lysis Buffer, from different types of sample materials of mammalian origin (such as urine, BAL, sputum, CSF, and swabs), pretreatment with MagNA Pure Bacteria Lysis Buffer (BLB) is required. The total input volume, including BLB, is 400 μl.

1.5. Preparation Time

Assay Time• Setup: 15 minutes• Total time to purify 8 blood samples: 25 minutesAdditional hands-on time is required for manual pre-isolation steps of the optional bacterial DNA isolation workflow.

No hands-on time is required after setup.



2. How to Use this Product



2.1. Before you Begin

Sample MaterialsTo obtain optimal results in downstream applications, such as real-time PCR assays on the LightCycler® 480 and LightCycler® Carousel-Based Instruments, follow the Instructions for Use. Do not process samples with higher volume or cell count than the selected purification protocol is designed to handle. This will lead to reduced yield since the magnetic glass beads will clump and not bind DNA as efficiently when too much high viscosity DNA is present.

Treat all samples as potentially infectious.

Optimal amounts of sample material are as follows:

• 100, 200, 300, or 400 μl of mammalian whole blood, containing no more than 5 × 103 WBCs/μl.If you use lower volumes of whole blood samples as indicated above, add PBS to the next possible volume and mix well. All cell numbers given in this protocol were determined manually with a standard hemocytometer. For best results, determine the number of WBCs in whole blood samples manually with a hemocytometer (such as a Neubauer device with counting chambers) before processing. Please note that automated counting systems sometimes produce cell counts that are not in agreement with cell counts produced by a manual hemocytometer.

The purification protocols for whole blood were established with human blood samples from healthy individuals. Unhealthy or drug-treated individuals may show abnormal blood cell numbers that can affect the nucleic acid isolation process.

Different mammalian species may have different concentrations of blood cells. For some species, you may need to use a smaller sample of blood to keep the cell numbers within the above guidelines. If necessary, test various amounts of blood to determine the volume that produces the highest, most reproducible yield.

Use only whole blood containing anticoagulants. Whole blood may be stored at +15 to +25°C for a maximum of one day, or at +2 to +8°C for one week. For longer storage times, freeze whole blood samples to prevent a gradual reduction in DNA yield and DNA integrity. 100, 200, 300, or 400 μl suspension of mammalian blood cells or buffy coat in PBS, containing no more than 5 × 103 WBCs/μl).• 100, 200, 300, or 400 μl of mammalian serum or plasma.• Up to 1 × 106 cultured cells in a volume of 100 μl PBS.

Centrifuge the cells to remove culture medium, then resuspend the cell pellet in 100 μl PBS. The DNA content of different cell lines may vary to a large extent due to different degrees of aneuploidy. First reduce the number of cells and use 2 to 5 × 105 cells for cell lines with an extremely high DNA content (e.g., K-562 or HeLa cells) to avoid clumping.For the isolation of bacterial DNA from different types of sample materials of mammalian origin (such as urine, BAL, sputum, CSF, and swabs), in combination with the MagNA Pure Bacteria Lysis Buffer, the input sample volume is 200 μl after pretreatment (including MagNA Pure Bacteria Lysis Buffer; BLB) it is 400 μl.





Control ReactionsAlways run appropriate controls with the samples, especially if you want to perform quantification analyses of the eluted nucleic acid samples (e.g., by real-time PCR assays on the LightCycler® Instrument). In order to control the complete process starting from sample preparation to quantification analysis, perform the following controls:

• Positive Control, by using a sample material positive for your target.• Negative Control, by using a sample material negative for your target.• Internal Control (IC), by adding a defined amount of a control template to all samples to be purified.

The Internal Control (IC) is added prior to the purification step and then co-purified and amplified with your target of interest from the specimen in the same PCR reaction. The IC concept is especially useful for enzyme-based amplification processes such as PCR, because efficiency of the PCR process might be reduced by inhibitors present in the purified sample material. In addition, the Internal Control is used to compensate for possible losses of your target during purification. For PCR quantification assays on the LightCycler® instruments, use a synthetic double-stranded DNA molecule with primer-binding sites identical to those of your target sequence, but having a unique probe-binding region that differentiates the IC from the target-specific amplicon. For RT-PCR assays, use an in vitro-transcribed RNA molecule with the same features as above. Discriminate the signals derived from your target and the IC by performing a dual-color HybProbe assay. For detailed information regarding the IC concept in combination with the LightCycler® Carousel-Based System, read the LightCycler® Technical Note 12/2000 “Absolute Quantification with External Standards and an Internal Control” available at www.lightcycler.com.

Internal ControlThe MagNA Pure Compact Instrument offers the option to add an Internal Control (IC) to the samples during the purification procedure.

Including an Internal Control is important to detect a possible impairment of the nucleic acid isolation process.

Two Possible Methods for Incorporating the IC:1. IC is added directly to the sample.• On Sample Ordering Screen I, set “Internal Control Volume” to “none”. Document the use of the IC in the

“Comment” field.• Use this method for suspensions of cultured cells in PBS.

Do not use this method when the sample is blood or plasma/serum and the IC is naked DNA or RNA, because the IC might be degraded by nucleases present in these sample materials.

2. IC is provided in the Tube Rack• Pipette the IC (5, 10, or 20 μl) into a separate tube. Use only the tubes specified in “Additional Equipment and

Reagents Required”.• Place each tube in the assigned position in row 2 of the Tube Rack.• On Sample Ordering Screen I, enter the amount of IC used.• The instrument automatically incorporates the IC into the isolation process, mixes it with lysis buffer, thereby,

protecting it from degradation by nucleases.• Use this method for whole blood, serum, or plasma samples. Naked DNA or RNA can be used as IC.

If you chose the IC option in the software and you do not place an IC tube in the Tube Rack, lysis buffer will be pipetted onto the instrument stage and cleaning of the instrument will be necessary. If your MagNA Pure Compact Instrument uses Software version 1.1, the lysis buffer might come into contact with the sample before it is pipetted into the IC tube. In this case, cleaning and decontamination of the instrument is necessary.





General Considerations

PrecautionsThe following procedure is designed to process 8 samples at the same time. The MagNA Pure Compact can handle all numbers of samples between 1 and 8. For a detailed description of instrument setup and handling, refer to the MagNA Pure Compact Operator’s Manual.

Adapt the Reagent Cartridge to +15 to +25°C before use. If you use the reagents at temperatures outside the recommended range, the kit may not work well. To ensure correct pipetting, use only the MagNA Pure Tube 2.0mL contained in the kit and the recommended types of tubes for the Internal Control (See Section Additional Equipment and Reagents Required. Document the lot number of the kit in case of complaints or questions for Roche Technical Services regarding any component of the kit (reagent cartridges or disposables). Make sure you have followed the instructions regarding type and amount of sample material. Wrong type and amount of sample material may cause clumping of MGPs which cannot be detected by the Clot Detection function of the instrument. Clumping of MGPs may lead to low yield and purity of nucleic acids, cross-contamination, and inhibition of downstream assays (e.g., PCR). After the run has finished, carefully inspect the instrument for any signs of spillage. If spillage occured, clean the instrument as described in the Operator’s Manual.Some buffers (wells 2, 4, and 5) contain guanidinium salts which are hazardous irritants. Do not allow these to come into contact with skin, eyes, or mucous membranes. If contact does occur, wash the affected area immediately with large amounts of water. If necessary, immediately contact your laboratory supervisor and seek medical assistance. When spilling these reagents, dilute the spill with large amounts of water before attempting to clean up the spill. Do not allow reagents containing guanidine thiocyanate (well 2) to contact sodium hypochlorite (bleach) solution or acids. These mixtures produce a highly toxic gas.

Prevention of Cross-Contamination• To minimize the risk of cross-contamination and to prevent contact with potentially infectious materials, always

complete the processes involving sample preparation such pipetting of blood samples in a designated part of the benchtop or safety hood before starting PCR setup.

• To further minimize the risk of contamination, always carry out sample preparation, PCR setup, and the PCR run itself in separate rooms specially designated for each phase in the workflow.

Safety Information

Laboratory Procedures• Handle all samples as if potentially infectious, using safe laboratory procedures. As the sensitivity and titer of

potential pathogens in the sample material varies, the operator must optimize pathogen inactivation by the Lysis/Binding Buffer or take appropriate measures, according to local safety regulations.

• Do not eat, drink or smoke in the laboratory work area.• Do not pipette by mouth.• Wear protective disposable gloves, laboratory coats, and eye protection when handling samples and kit reagents.• Do not contaminate the reagents with bacteria, viruses, or nucleases. Use disposable pipettes and nuclease-free

pipette tips only to remove aliquots from reagent bottles. Use the general precautions described in the literature.• Wash hands thoroughly after handling samples and reagents.• Complete each phase of the PCR/RT-PCR workflow before proceeding to the next phase. For example, you should

finish PCR/RT-PCR sample preparation before starting PCR/RT-PCR setup. Sample preparation, PCR/RT-PCR setup and the PCR/RT-PCR run itself should also be performed in separate locations.

Waste Handling• Discard unused reagents and waste in accordance with country, federal, state, and local regulations.• Safety Data Sheets (SDS) are available upon request from the local Roche office.





2.2. ProtocolsPre-isolation steps are required for DNA isolation from blood cells (WBCs, PBMCs, buffy coat), and for the DNA_Blood_external_lysis and Total_NA_Plasma_external_lysis purification protocols, which include a manual sample lysis step. The protocols were developed with and for human blood or serum/plasma. Always prepare lysates freshly and process them immediately. Store the lysates at −70°C or below when nucleic acid isolation is postponed.

External Lysis ProtocolFor external lysis of samples, follow the procedure below:

Transfer 100, 200, 300, or 400 μl of whole blood or serum/plasma sample into a suitable vial.

Add 300 μl MagNA Pure LC DNA Isolation Kit I – Lysis/Binding Buffer Refill*.

Mix the samples thoroughly by pipetting. To ensure complete inactivation of the sample, incubate the lysate for up to 30 minutes on a roller incubator.

Transfer the sample lysate (400, 500, 600, or 700 μl) into the Sample Tube.

Place the MagNA Pure Tube 2.0mL in the Tube Rack and start the DNA_Blood_external_lysis or Total_NA_Plasma_external_lysis purification protocol.

Blood CellsFor isolation of DNA from blood cells, perform the following pre-isolation steps:

• White Blood Cells (WBCs): Lyse red blood cells of EDTA-preserved blood with Red Blood Cell Lysis Buffer*. Harvest the WBCs by several centrifugation steps according to the Instructions for Use. Adjust WBCs in PBS to a cell number of 5 × 105 cells/100 μl.

• Peripheral blood mononuclear cells (PBMCs): isolate PBMCs from whole blood in Vacutainer CPT tubes (Becton-Dickinson) by several centrifugation steps according to the instructions of the tube supplier. Adjust PBMCs in PBS to a cell number of 5 × 105 cells/100 μl.

• Buffy Coat: Dilute with an equal amount of PBS prior to use. 400 μl of the buffy coat/PBS suspension should not contain more than 2 × 106 cells.

Purification Protocols from Bacterial-Containing SourcesFor the MagNA Pure Compact Instrument plus MagNA Pure Compact Nucleic Acid Isolation Kit I: The input sample volume is 200 μl; after pretreatment (including MagNA Pure Bacteria Lysis Buffer; BLB), the sample volume is 400 μl. The DNA_Bacteria purification protocol can adjust the volume of Elution Buffer to 50 μl, 100 μl, or 200 μl. Use an elution volume of 50 μl for liquid samples of low cell content (e.g., urine) only.

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Overview of Treatments For Bacterial Sample Materials With MagNA Pure Bacteria Lysis Buffer

MagNA Lyser Treatment

This is an optional step for automated mechanical homogenization. This step is very useful for samples containing particles, such as stool. It also enhances the lysis of gram-positive bacterial species.This is an optional step for automated mechanical homogenization.

Transfer sample (up to 500 μl) to a MagNA Lyser Green Beads tube.

Place the tube in the MagNA Lyser Instrument.

Homogenize for 30 seconds at 6,000 rpm.

Cool samples for 1 minute in the MagNA Lyser Rotor Cooling Block.

Centrifuge 5 minutes at 17,000 × g at +15 to +25°C.

Transfer 400 μl of the lysate supernatant into an appropriate tube.

For some types of sample, you may improve DNA yield by performing this homogenization step first, before adding Bacteria Lysis Buffer.

Liquefaction

This step is for very viscous samples, such as BAL or sputum.Prepare a fresh DTT stock solution (e.g., 5x concentrated = 0.75%).

Adjust the final concentration of DTT in the sample to 0.15% by adding appropriate amounts of DTT stock solution.

Incubate the sample by shaking for 30 minutes at +37°C, until it can be easily pipetted.

Centrifugation

This is an optional step for large volume liquid samples that have low or unknown bacterial loads, such as urine, CSF, BAL, or aspirates.

Centrifuge the sample for up to 10 minutes at 20,000 × g to concentrate the bacterial cells in the pellet.

Discard most of the supernatant leaving only 200 μl.

Resuspend the cells in this remaining liquid, then process this concentrate.

This centrifugation step allows a dilute sample that originally contains several milliliters of liquid to be processed in a single isolation, ensuring maximum DNA yield.

Addition of Bacteria Lysis Buffer

Add 180 μl of MagNA Pure Bacteria Lysis Buffer (BLB) to 200 μl of sample. – When the sample volume is less than 200 μl, add enough BLB to make a final volume of 380 μl.

When the MagNA Lyser is used to homogenize the sample, increase the volume of both the sample and BLB to 250 μl each. Final volume, BLB plus sample = 500 μl.

Mix well.

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Enzymatic Digestion

This is an optional step to enhance lysis of certain bacteria, such as gram-positive species.Depending on the type of bacteria to be lysed, prepare one of the following enzyme cocktails: – Enzyme Cocktail I: N-acetylmuramidase (0.625 mg/ml = 2.500 U/ml) and beta-1,3-glucanase (Zymolyase) (0.25 mg/ml = 500 U/ml); dissolved in 50% glycerol/50 mM Tris-acetate, pH 6.7. This cocktail lyses a broad spectrum of gram-positive bacteria. – Enzyme Cocktail II: lysozyme (100 mg/ml = 50 kU/ml) and lysostaphin (5 mg/ml = 5 kU/ml); dissolved in 5% glycerol/PBS, pH 7.5. This cocktail is especially useful for lysis of Staphylococcus spec.

Add 10 μl (per 400 μl total sample volume of the enzyme cocktail) to the BLB/sample mixture.

You can premix Enzyme Cocktail and BLB and add them both to the sample in a single step.

Incubate at +37°C for 10 to 30 minutes.

Proteinase K Digestion

Add 20 μl of Proteinase K to the mixture and mix thoroughly.

If you do not use an enzyme cocktail, premix Proteinase K and BLB and add them to the sample in a single step.When the MagNA Lyser is used to homogenize the sample (see above), increase the volume of both the sample and BLB/Proteinase K to 250 μl each. Final volume of BLB, plus Proteinase K, plus sample = 500 μl.

Incubate for 10 minutes at +65°C (e.g., BALs) or overnight at +15 to +25°C for biopsies or solid tissue until the sample is completely dissolved and can be resuspended.

Boiling

For best results, perform this step to inactivate pathogenic organisms in the sample. It may also enhance lysis of the cell wall of some bacterial species. To prevent leakage, perform this step in screw-capped reaction tubes.Not recommended for isolation of RNA because this could negatively effect the integrity of the RNA.Incubate samples at +95°C for 10 minutes.

Recentrifuge briefly to collect the complete sample volume at the bottom of the tube.

Allow samples to cool down or chill on ice, then transfer the cooled sample to the MagNA Pure Tube 2.0mL.

Special Sample Materials

StoolTake a peanut-sized amount of stool sample and suspend it in 200 μl PBS.

For best results, homogenize stool samples with the MagNA Lyser Instrument to completely dissolve solid particles.To avoid clogging the reaction tips with undissolved solid particles, sediment solid particulates in the lysate by centrifuging briefly at 500 × g.

Transfer the clarified supernatant to a fresh tube.

Add 180 μl BLB and 20 μl of Proteinase K.

Incubate for 10 minutes at +65°C, followed by 10 minutes at +95°C.

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BiopsiesAdd an appropriate volume of BLB and Proteinase K (e.g., 180 μl and 20 μl, respectively) to a 1 to 10 mg piece of tissue.

Incubate at +65°C until the sample is completely dissolved and can be resuspended.

Incubate for 10 minutes at +95°C.

Transfer lysate (up to 400 μl) to the MagNA Pure Tube 2.0mL.

SwabsSuspend the swab in an appropriate volume of BLB and proteinase K (e.g., 200 μl and 20 μl, respectively).

Squeeze the liquid from the swab and collect the liquid.

Incubate at +65°C for 10 minutes, followed by 10 minutes at +95°C.

Transfer lysate (up to 400 μl) to the MagNA Pure Tube 2.0mL.

Purification ProtocolTo perform DNA isolation from mammalian blood or cultured cells, or total nucleic acid isolation from mammalian serum/plasma samples with the MagNA Pure Compact Nucleic Acid Isolation Kit I, different pre-installed purification protocols are available. For each protocol, sample and elution volumes must be chosen from the software menu. For DNA isolation from mammalian blood or total nucleic acid isolation from serum/plasma samples in combination with external lysis of the primary samples, two additional purification protocols are available:• DNA_Blood_external_lysis• Total_NA_Plasma_external_lysis.Using these protocols, samples are lysed manually, outside the MagNA Pure Compact Instrument. Lysates are then transferred to the instrument and purification is carried out automatically. This procedure allows to physically separate the lysis step from the purification step and to load inactivated sample material to the MagNA Pure Compact Instrument (e.g., when using potentially infectious sample material). For isolation of bacterial DNA with the MagNA Pure Compact Nucleic Acid Isolation Kit I, the MagNA Pure Compact Instrument uses the DNA_Bacteria purification protocol. New protocols or protocol updates can be downloaded from http:\www.magnapure.com. Read the instructions for downloading and installing the purification protocol carefully. For additional details, contact your local Roche representative. Use the following table to decide which purification protocol is best for your sample material:

The different protocols are optimized for a specific sample material each. Do not use a protocol for a sample material other than specified below. Doing so will affect the performance of the isolation process, may lead to clumping and loss of MGPs, cross contamination of samples, or even damage to the instrument

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Onboard MagNA Pure Compact software programs for six purification protocols currently available for use with the MagNA Pure Compact Nucleic Acid Isolation Kit I:

Protocol Name Sample Material Optional Internal Control 1) Elution Volume 3) DNA_Blood_100_400 • 100, 200, 300, or 400 μl whole blood

(≤5 × 103 cells/μl)yes 100 or 200 μl

DNA_Blood_external_lysis • 400, 500, 600, or 700 μl yes 100 or 200 μl Total_NA_Plasma_100_400 • 100, 200, 300, or 400 μl plasma or

serumyes 50 or 100 μl

Total_NA_Plasma_external_lysis

• 400, 500, 600, or 700 μl lysate (serum/plasma + lysis buffer)

yes 50 or 100 μl

DNA_Culture_Cells • 100 μl cell suspension (≤1 x 106 cells)

no 2) 200 μl

DNA_Bacteria • 400 μl sample lysate in MagNA Pure Bacteria Lysis Buffer

yes 50, 100, or 200 μl

Only one protocol (and thus one sample material) can be selected for each run. Therefore, different types of sample material (or different sample or elution volumes) cannot be combined in the same run.

1) see “Internal Control” for detailed information. 2) The DNA_Culture_Cells protocol does not allow to use an Internal Control. If an Internal Control is required in combination with this protocol, pipette approximately 20 μl Internal Control directly into the cell suspension (the Internal Control is stable in PBS).3) The concentration of nucleic acids in the eluate, and therefore sensitivity in downstream applications can be increased by choosing a low elution volume; however, elution efficiency and overall nucleic acid yield will be approximately 10 to 30% less compared to the high elution volume (e.g., the typical DNA yield from 100 μl human whole blood using an elution volume of 100 μl is 5.9 μg, compared to 7.9 μg when using 200 μl elution volume). For DNA isolation from cultured cells, this variable elution volume option is not included due to the very high DNA content of this sample material.





MagNA Pure Compact Protocol Using the MagNA Pure Compact Nucleic Acid Isolation Kit IIsolate nucleic acids according to the protocol below:

Turn on the instrument. – Remove Cartridge Rack and Tube Rack (with Elution Tube Rack) from the instrument. – Click the Run button on the Main Menu Screen to access Sample Ordering Screen 1. – Follow the software-guided workflow.

Remove a prefilled Reagent Cartridge from the blister pack.

Check the cartridge integrity and filling volumes of the wells. Do not use cartridges that have a different pattern of filling or that are damaged.

– Adapt the Reagent Cartridge to +15 to +25°C for 30 minutes.Handle each Reagent Cartridge prior to use as follows:

– Always wear gloves when handling the MagNA Pure Compact Cartridge. – Hold the cartridge only at the barcode imprinted area and the opposite side. – Avoid touching the sealing foil covering the cartridge wells. – Avoid touching the two single open wells and do not use them as handles. – Avoid any foam formation and let the fluid within the cartridge wells settle again completely. If fluid remains under the sealing foil, knock the cartridge bottom gently on a flat lab bench surface. This is especially important for well 1 which contains a small volume of Proteinase K.

Scan the barcode. – With the two isolated wells pointing away from you, insert all the wells on the Reagent Cartridge into the holes in the Cartridge Rack. – Use the guide slots on the rack to help position the cartridge. – Repeat the steps above for the desired numbers of samples (1 to 8).

Proceed to Sample Ordering Screen 2. – Select the appropriate purification protocol from the Protocol menu. – Select the elution volume (50 μl, 100 μl, or 200 μl). – Optional: Select the Internal Control Volume (5, 10, or 20 μl).

Insert the appropriate number of Tip Trays (one per purification) into the assigned position in the instrument Tip Rack.

Check if the Tip Tray holds a disposable in each position (tip or piercing tool). Do not use Tip Trays that are not assembled accordingly.Handle Tip Trays with care to prevent tips or piercing tool from falling out of the tray. Should this happen, discard the respective Tip Tray and tips. Use the Tip Tray Kit to replace missing Tip Trays.

– Proceed to Sample Ordering Screen 3.

Scan the sample barcode from the primary sample tube or enter the sample name. – Arrange the Sample Tubes (MagNA Pure Tube 2.0mL) in row 1 of the Tube Rack. Make sure the brim of the tubes seats solidly on the rack. – Pipette the samples into their respective Sample Tubes. – Proceed to Sample Ordering Screen 4.

This screen only appears and accepts information about Internal Control Tubes if you selected a protocol with internal controls on Sample Ordering Screen 2. The program will skip this screen and proceed directly to Sample Ordering Screen 5 if you selected a protocol with no IC.

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Pipette the proper amount of IC (as specified on Sample Ordering Screen 2) into one of the IC Tubes. – Identify the tube by attaching your own barcode label or writing an ID number on the tube with a permanent marker. – Put the filled control tubes in row 2 of the Tube Rack. –Touch IC Sample ID field 1. In that field, enter the ID of Control 1 (i.e., the one you just put in the rack) by either scanning the (self-attached) barcode or using the virtual keyboard to type the ID number. (Touch the Keyboard button to access the keyboard). – Repeat the above control placement and identification steps for each control used. – Insert the Tube Rack with sample and ICs. – Touch the IC Tubes inserted confirmation button.

Reinsert the Tube Rack into the instrument. – Proceed to Sample Ordering Screen 5.

Scan the barcodes of the Elution Tubes ( MagNA Pure Tube 2.0mL). – Place the Elution Tubes into the Elution Tube Rack. Make sure the brim of the tubes seats solidly on the rack. – Reinsert the Elution Tube Rack into the instrument.

On the Confirmation Screen, check the information display. – If the information is correct, confirm it by touching the Confirm Data button, close the front cover, and start the run.

After the purification run has ended, the Result Screen appears, showing the result of the isolation process for each channel: – The result will be PASS if the isolation run was completed without any warning or error. – The result will be FAIL if any interruption of the process or error occurred during the run. For each FAIL result, the result screen will show a brief error or warning messages to help you decide whether the error or warning can be ignored. Refer to the troubleshooting section of the MagNA Pure Compact Operator’s Manual.

Close the MagNA Pure Tubes 2.0mL containing eluates with the supplied tube caps and remove the Elution Tube Rack or the tube immediately after the end of the purification run. – If not proceeding directly to your downstream application, store eluates at −20°C (if target nucleic acid is DNA) to −70°C (if large nucleic acid is RNA). Nucleic acids are stable for at least 6 to 12 months if stored properly.

Optionally: Start the automated liquid waste discard. – Always empty the MagNA Pure Compact Waste Tank after every purification run. – Treat liquid waste as potentially infectious (depending on sample material), and hazardous, since lysis buffers are present (see section SafetyInformation).

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3. Results


3. ResultsDNA Yield and PurityDNA was isolated from normal human whole blood samples or from K-562 cultured cells, and eluted in 200 μl Elution Buffer. Yields were determined by OD260 nm measurement. Purity was determined by OD260 nm/280 nm measurement. For exemplary results, see table below. Sample Material Volume/Amount DNA Yield [μg] OD260/280 ratio.

Sample Material Volume/Amount DNA Yield (μg) OD260 nm/280 nm RatioWhole blood (containing 8,700 cells/μl) 100 μl 5.3 ≥1.9

200 μl 9.6 ≥1.9Whole blood (containing 5,000 cells/μl 100 μl 3.6 ≥1.9

200 μl 6.2 ≥1.9300 μl 6.6 ≥1.9400 μl 10.3 ≥1.9

K-562 cells 5 × 105 8.3 ≥1.95K-562 cells 1 × 106 19.5 ≥1.95

DNA yield from whole blood is dependent on the blood donor and blood cell count. The yield from cultured cells is dependent on the degree of aneuploidy of the cell line. Plasma/serum samples do not contain enough DNA to be measured by OD260 , but the amounts purified can be measured using real-time PCR with LightCycler® 480 and LightCycler® Carousel-Based Instruments.

DNA IntegrityDNA was isolated in replicates of 8 from human whole blood taken from two different donors. Elution volume was set to 200 μl. DNA integrity was shown using agarose gel electrophoresis with a Molecular Weight Marker III. All samples showed genomic DNA bands with a size of at least 20 kb.

Fig. 2: 2.5 μl DNA isolated from 200 μl human whole blood analyzed by agarose gel electrophoresis



3. Results


SensitivityA dilution series of human Parvo B19 Virus in the range of 102 to 107 copies/ml was added to a citrate-preserved plasma solution. Samples (200 μl) of this spiked plasma were used for nucleic acid isolation and harvested in an elution volume of 100 μl. LightCycler® HybProbe PCR assay targeting Parvo B19 virus showed that 100 copies/ml were easily detected in 5 μl of eluate, demonstrating the outstanding sensitivity and linearity of the MagNA Pure Compact Nucleic Acid isolation Kit I procedure.

Fig. 3: LightCycler® HybProbe analysis using PCR (targeting Parvo B19 Virus) of DNA samples isolated from whole blood samples spiked with different concentrations of Parvo B19 Virus.

ScalabilityDNA isolated from samples with increasing amounts of whole blood was amplified using real-time PCR on the LightCycler® 2.0 Instrument using the LightCycler® HER2/neu DNA Quantification Kit (Roche Molecular Diagnostics). The resulting crossing points confirm the excellent scalability of the isolation procedure. No signs of PCR inhibition were observed. Due to the nature of the PCR, there is a linearity of crossing points versus the logarithm of template amount.

Fig. 4: LightCycler® HybProbe analysis using PCR (targeting Her2/neu) of DNA samples isolated from different whole blood volumes.



4. Troubleshooting


4. TroubleshootingObservation Possible cause RecommendationClumping of beads/problem with magnetic separation of beads.

Too much sample material. Reduce amount of sample material to the values recommended in section Sample Materials.

MGPs were magnetized prior to use. Avoid contact between MGPs and magnets. Store kit appropriately.

Carryover of MGPs into eluate.

Too much sample material was used causing clumping of MGPs.

Reduce amount of sample material to the values recommended in section Sample Materials.Centrifuge MagNA Pure Tube 2.0mL to sediment MGPs. Transfer MGP-free supernatant to a fresh tube.

Nucleic acid is degraded. Storage of samples was not optimal. Use fresh or frozen samples. Avoid using samples that were stored at +15 to +25°C for extended periods of time.

Storage of samples was not optimal. Do not store eluates at +15 to +25°C.Do not perform overnight runs.

Nuclease contamination of Reaction Tips, Sample Tubes, Elution Tubes, or reagents.

Avoid contaminating disposables and reagents with nucleases.

Poor nucleic acid yield. Sample did not contain enough cells. Cultured cells: Count cells before use. Optimal results are obtained with 5 × 105 cells.Blood: Do not use clotted or sedimented blood. Use fresh or frozen blood containing anticoagulants. Mix tubes before use.

Storage of samples was not optimal. See above.Too much sample material was used causing clumping of MGPs.

Reduce amount of sample material to the values recommended in section Sample Materials.

Reagents were in wrong position on stage.

Make sure that all reagents are in their correct positions on the stage.

Poor nucleic acid purity Too much sample material. Reduce amount of sample material to the values recommended in section Sample Materials.

Storage of samples was not optimal. See above.Cross-contamination of samples.

Too much sample material was used. Reduce amount of sample material to the values recommended in section Sample Materials.



4. Troubleshooting


Observation Possible cause RecommendationPoor performance in downstream assays (PCR/RT-PCR).

Concentration of template DNA was not optimal.

Check DNA concentration in the eluted DNA samples and adjust input amount of eluate per PCR.Optimum: 10 – 100 ng DNA/PCR.Optimize amount of input sample material. Refer to the section Sample Materials.

Poor purity of DNA. Too much sample material. Refer to the section Sample Materials.

Storage of samples was not optimal. See above.Improper storage of eluate. See above.(RT-) PCR reagents or protocols were not optimal.

Check (RT-)PCR reagents and protocols with a positive DNA or RNA control (e.g., Human Genomic DNA*) or use an Internal Control.

Eluates show slightly red color.

Minimal abrasion from magnetic particles.

Centrifuge at low g-values (approximately1,000 rpm) to remove fines.

The red color does not affect subsequent (RT)-PCR assays on the LightCycler® Instruments or conventional thermal block cycler.

Low yields of elution volume. In some cases, only a certain portion of the eluted material is transferred to the elution tubes.

The quality of the isolated nucleic acid is not impacted. We recommend manually transferring that portion of the eluate still remaining in the reagent cartridge to a vial either for storage or a subsequent application, such as PCR.



5. Additional Information on this Product


5. Additional Information on this Product

5.1. Test Principle

How this Product WorksMagNA Pure Compact Nucleic Acid Isolation Kit I is used with the MagNA Pure Compact Instrument to purify high-quality, undegraded genomic DNA from mammalian blood, blood cells, buffy coat, or cultured cells, and total nucleic acids (e.g., viral DNA and RNA) from mammalian serum or plasma, using the MagNA Pure Compact Instrument. The isolated DNA can be eluted into 50, 100, or 200 µl (depending on the purification protocol used). It meets the quality standards required for highly sensitive and quantitative PCR or RT-PCR analysis on the LightCycler® Instruments. The MagNA Pure Compact System consists of the instrument, reagents, and disposables:

• The instrument can perform 1 to 8 isolations of DNA per run.• The isolation reagents are provided in prefilled, sealed Reagent Cartridges. Each cartridge contains all reagents

required for one isolation.• The reaction tips needed for each isolation are provided in a disposable Tip Tray. Also, barcoded and sterile tubes

for uptake of samples and nucleic acid eluates are supplied.• In combination with the MagNA Pure Bacteria Lysis Buffer* and the DNA_Bacteria purification protocol, bacterial

DNA can be isolated from different types of sample of mammalian origin (such as urine, BAL, sputum, CSF, swabs) or bacterial cultures.

After instrument setup and starting the software-guided isolation protocol, the MagNA Pure Compact Instrument performs all isolation steps automatically.

Test PrincipleThe nucleic acid isolation procedure is based on the proven MagNA Pure magnetic-bead technology. The principal steps of the subsequent MagNA Pure Compact nucleic acid isolation procedure are:

The samples are lysed by incubation with Proteinase K and a special lysis buffer containing a chaotropic salt.

Magnetic Glass Particles (MGPs) are added and nucleic acids are immobilized on the MGP surfaces.

Unbound substances such as proteins, cell debris, and PCR inhibitors are removed by several washing steps.

Purified nucleic acids are eluted from the MGPs.

5.2. Quality ControlThe MagNA Pure Compact Nucleic Acid Isolation Kit I is function tested using several model systems: Genomic DNA is isolated from human whole blood. Quality of purified DNA is checked by agarose gel electrophoresis and a real-time PCR assay on the LightCycler® Carousel-Based Instrument targeting Cyclophilin A.• Total nucleic acid is isolated from human plasma spiked with DNA from Parvovirus B19, Hepatitis A Virus (HAV), or

Phocine Herpesvirus 1 (PhHV-1).• Quality of purified total nucleic acid is checked by real-time PCR assays on the LightCycler® Carousel-Based

Instrument targeting Parvovirus B19 or HAV and on the ABI PRISM Sequence Detection System 7700 targeting PhHV-1.

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6. Supplementary Information



6.1. ConventionsTo make information consistent and easier to read, the following text conventions and symbols are used in this document to highlight important information:

Text convention and symbolsInformation Note: Additional information about the current topic or procedure.Important Note: Information critical to the success of the current procedure or use of the product.

. .etc. . etc.

Stages in a process that usually occur in the order listed.Steps in a procedure that must be performed in the order listed.

* (Asterisk) The Asterisk denotes a product available from Roche Diagnostics.

6.2. Changes to previous versionLayout changes. Editorial changes. See Kit Contents wording changes: Replaced Sample and Elution Tube with MagNA Pure Tube 2.0mL. Replaced Elution Tube Cap with MagNA Pure Tube Cap.

6.3. Ordering InformationRoche offers a large selection of reagents and systems for life science research. For a complete overview of related products and manuals, please visit and bookmark our homepage lifescience.roche.com.

Product Pack Size Cat. No.InstrumentsMagNA Pure Compact Instrument 1 instrument with integrated PC,

touchscreen monitor and bar-code reader03 731 146 001

Reagents , kitsMagNA Pure Compact RNA Isolation Kit 1 kit, 4 x 8 sealed cartridges, 32 isolations 04 802 993 001MagNA Pure DNA Tissue Lysis Buffer 1 bottle, 100 ml 04 805 160 001MagNA Pure Compact Nucleic Acid Isolation Kit I – Large Volume

1 kit, 4 x 8 sealed cartridges, 32 isolations 03 730 972 001

MagNA Pure Compact Tip Tray Kit 10 tip trays 03 753 166 001

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Roche Diagnostics GmbHSandhofer Strasse 11668305 MannheimGermany


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6.4. Trademarks HYBPROBE, LIGHTCYCLER, MAGNA LYSER and MAGNA PURE are trademarks of Roche. All third party product names and trademarks are the property of their respective owners.

6.5. License DisclaimerFor patent license limitations for individual products please refer to: http://technical-support.roche.com.

6.6. Regulatory DisclaimerFor general laboratory use.

6.7. Safety Data SheetPlease follow the instructions in the Safety Data Sheet (SDS).

6.8. Contact and SupportIf you have questions or experience problems with this or any Roche product for Life Science, please contact our Technical Support staff. Our scientists are committed to providing rapid and effective help.Please also contact us if you have suggestions for enhancing Roche product performance or using our products in new or specialized ways. Such customer information has repeatedly proven invaluable to the research community worldwide.

To ask questions, solve problems, suggest enhancements or report new applications, please visit our Online Technical Support Site.

Visit lifescience.roche.com, to download or request copies of the following Materials:• Instructions for Use• Safety Data Sheets• Certificates of Analysis• Information Material

To call, write, fax, or email us, visit lifescience.roche.com and select your home country to display country-specific contact information.

http://technical-support.roche.com

http://technical-support.roche.com

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