AnaPrep Automated DNA and RNA Purification System

AnaPrep
image source: www.biochain.com

Overview

BioChain’s AnaPrep system is a fully automated nucleic acid extraction platform that is efficient, effective and economical. Based on the latest magnetic bead technology, AnaPrep is easy to use with three simple steps: load samples, run pre-programmed protocols, and collect purified samples. The end result is reproducible high quality nucleic acids from a wide range of starting materials that are ready for a variety of downstream applications.

Features/ Benefits:

• Automated processing of 1 to up to 24 samples with pre-programmed protocols
• Consistent high purity nucleic acid using reliable reagents and methods
• Saves time and labor with pre-filled reagent cartridges and disposables
• Flexible design for processing customized sample numbers and elution volume (50-300ul).
• For research use only in the US, manufactured under GMP, ISO13485, and CE marking
• Minimizes contamination risks with unidirectional sample head and built-in UV lamp
• Versatile system enables purification from various sample sources and the products are suitable for many downstream applications
• Easy to Use and Error-free with simple steps, fool-proof design, and barcode reader
• Conserves space with an all-in-one, compact instrument 

Principle

The efficiency of AnaPrep stems from making the extraction process easy. 

AnaPrep combines the latest magnetic bead separation technique with strict quality control systems for reagents and liquid handling to obtain pure extraction. Researchers save time, labor, and avoid mistakes by using pre-filled reagent cartridges and disposable plastics. The purification process is as fast as 40 minutes for 1-12 or 24 samples. 

With an integrated processing unit that carries out the preloaded protocols, AnaPrep eliminates the requirement for an external computer. AnaPrep comes with a barcode reader enabling quick selection of protocols. The seamless automation provides the user the ability to simply place the samples in the machine, select a pre-programmed protocol, walk away, and let the machine carry out the tedious steps required for extraction. All processing procedures are done by the instrument, from piercing the reagent cartridge to eluting the pure nucleic acid. Using the machine is easy and does not require training in automation. 

Specimen Application 

Versatility in any system affords users the resources to expand their work and AnaPrep is such a system. DNA from broad sample sources, such as blood, tissue, and FFPE have been successfully purified with AnaPrep extraction kits. The purified nucleic acids are immediately suitable for a broad range of downstream applications.

AnaPrep System is able to purify nucleic acid from the following biospecimen:

• Whole blood 
• FFPE
• Saliva
• Stool
• Serum 
• Tissue
• CSF
• Plasma
• Cell
• Urine
• Buffy Coat
• Swab
• Sputum 

Downstream Applications

• PCR
• qPCR
• Next generation sequencing
• Restriction enzyme digestion
• Hybridization
• SNP analysis
• Genotyping
• NAT, oncology, forensics, food inspection, GMO
• Molecular diagnosis (for applications sufficient with CE marking, or in LDT). For research use only in the US.

For detailed info, visit: biochain.com

Maxwell RSC- Automated DNA and RNA Purification with Integrated Quantitation

Overview

The Maxwell Rapid Sample Concentrator (RSC) Instrument is an automated nucleic acid purification system that will process up to 16 samples in a single run. Used in combination with prefilled reagent cartridges, the Maxwell RSC can purify DNA or RNA from a wide range of sample types. The intuitive graphical user interface makes the instrument easy to use, and the integrated Quantus Fluorometer lets you collect purification and quantification data in one report.

Specifications

• Processing Time: 30–60 minutes (depending on sample type and method)
• Number of Samples: Up to 16
• Weight: 24.2lb (11kg)
• Dimensions (W × D × H): 13 × 13.6 × 11.8 inches (330.2 × 345.2 × 299.7mm)
• Power Requirements: 100–240VAC, 50/60Hz, 2.5A
• Fuse: 250VAC, 2.5A, low breaking capacity, time-lag fuse (AC250V, T2.5AL, 5 × 20mm)
• UV Bulb: Average lifetime approximately 6,000 hours, length 135.9mm, diameter 16mm, 4W, 0.17A current, 29V, spectral peak F 253.7, UV output 0.9W

Instrument Standard Components

• 1 Maxwell RSC Instrument
• 1 Tablet PC preloaded with Maxwell RSC Application Software
• 1 USB Cable for connection of the Maxwell RSC Instrument to the Tablet PC
• 1 Power Cable for Maxwell® RSC Instrument
• 1 Power Cords for Tablet PC
• 1 Maxwell RSC/CSC Deck Tray
• 1 UV Bulb (installed)
• 1 Quantus™ Fluorometer and accessories
• 1 Tablet PC Holder
• 1 Quick Start Guide
• 1 Setup Guide

For more info, visit: promega.com

Karyotyping- Definition, Principle, Preparation and Technical Procedure

Definition and Principle

Karyotyping is a laboratory procedure that allows examination of a patient’s set of chromosomes. “Karyotype” also refers to the actual collection of chromosomes being examined. Examining these chromosomes through karyotyping allows your physician to determine whether there are any abnormalities or structural problems.

Chromosomes, which are in almost every cell of your body, contain the genetic material inherited from your parents. They are composed of deoxyribonucleic acid (DNA) and determine the way that every human being develops.

When a cell divides, it needs to pass on a complete set of genetic instructions to each new cell it forms. Normally, when a cell is not in the process of division, the chromosomes are arranged in a diffuse, unorganized way. However, during division, the chromosomes in these new cells line up in pairs. In a karyotype test, which examines dividing cells, these pairs are arranged by their size and appearance, allowing a doctor to easily determine if any chromosomes are missing or damaged.

Test Applications

If you have an unusual number of chromosomes, if they are arranged incorrectly, or if they are malformed, this can be a sign of a genetic condition, such as Down or Turner syndrome.

Karyotyping can be used to detect myriad genetic disorders. For instance, if a woman has premature ovarian failure, she may have a chromosomal defect that karyotyping can distinguish. The test is also useful for identifying the Philadelphia chromosome, the presence of which can signal chronic myelogenous leukemia.

Babies can be karyotype tested before they are born to diagnose genetic abnormalities that indicate serious birth defects, such as Klinefelter syndrome, in which a boy is born with an extra X chromosome.

Materials Needed

• Chromosome medium (one bottle per student)--- KaryoMAX Peripheral Blood Karyotyping Medium. These bottles usually come in groups of 10 (each bottle containing 5 mL of media).
• Colcemid solution (10 mL bottle) --- KaryoMAX Colcemid liquid
• Giemsa stain (100 mL bottle) --- KaryoMAX Giemsa Stain Stock
• Potassium chloride solution, 0.075M (about 10 mL per student) --- KaryoMAX Potassium 
Chloride Solution, 0.075M
• Sterile 1 or 5 mL syringe21-guage needle for the syringeGreen-top vacutube21-guage multidraw needle (for vactube)15 mL centrifuge tube
• Pasteur pipetteBulb for the pasteur pipette70 percent isopropyl alcohol pad

Equipments Needed

• Centrifuge
• Glacial acetic acid (ACS grade)
• Absolute methanol (ACS grade)
• Refrigerator
• Incubator (capable of 37oC)
• Frosted glass slides

Procedure

1. Preparation of cell culture

• Using a 21-guage multi-draw needle and a green top vacutube (both supplied by the local hospital, if you are lucky), a qualified technician withdraws blood from the students.
• Prepare a sterile 5 mL syringe with a 21-guage needle.
• Wipe the top of the green top tube [Bottle with needle] (containing blood) with an isoproponal alcohol pad.
• Insert the needle on the syringe into the green top and withdraw a few milliliters of blood.
• Open the bottle of chromosome medium and place five to ten drops of blood into the medium. Sterile technique must be used because it is possible to cause major contamination during this procedure. 

2. Incubation

• Mix the medium and blood by gentle inversion and place the bottle in a preheated incubator at 37o C.
• Incubation for 70 hours
• Mix gently by inversion twice a day during incubation 

3. Stopping the cell division at metaphase

• Pre-warm the Colcemid in the incubator at 37 degree Celsius. CAUTION: Colcemid can be dangerous, so handle with care. Colcemid is a mitotic spindle inhibitor. If splashed on skin, rinse immediately and seek medical help.
• Add 0.05 mL (50 microliters) of prewarmed 37oC Colcemid to the culture. Mix gently and put the culture back into the incubator.
• Incubate for 30 to 60 minutes. 

4. Hypotonic treatment of the red and white blood cells

• Remove the blood and Colcemid solution from the [Centrifuge] incubator and mix gently.
• Put the entire contents of the bottle into a conical centrifuge tube. If conical tubes are not available, regular tubes can be used.
• Centrifuge for six minutes at 500 - 900 rpm (see notes at the end of lab regarding centrifuge speed).
• After six minutes, turn off the centrifuge and wait for a complete stop. Carefully remove the tube.
• Remove the supernate (clearish fluid on top) with a pasteur pipette. Be very careful not to disturb the button of cells on the bottom. Make sure that the bulb of the pipette is depressed before it is inserted into the test tube. Leave some fluid (anywhere from * to * mL) on the top of the button of cells. When withdrawing fluid, keep the pipette tip against the side of the test tube to avoid any shaky movements.
• Add one mL of warmed 37oC hypotonic solution to the tube. Mix by flicking the tube with your finger. Now add another nine mL of hypotonic solution. The hypotonic solution should not be in contact with the cells for more than a total of 24 minutes. Excess exposure may cause rupture of the white blood cells.
• Throughly mix all the hypotonic fluid with the cells. This is done by drawing all the mixture at the bottom of the tube into a pasteur pipette and forcing it out again. Do this two or three times to thoroughly mix.
• Place the mixed solution into the 37oC incubator for nine minutes
• The fixative solution must be made fresh. While the hypotonic solution is working, make up the fixative solution as follows: add three parts chilled absolute methanol (or as close as you can get) to one part galacial acetic acid. Both chemicals should be as pure as possible.
• After nine minutes, centrifuge for six minutes at 500 to 900 rpm.
• Remove the supernate. leaving * or * mL of fluid on top of the button of cells. At this time you probably have a small whitish or reddish film at the bottom and slightly up the side of the tube. The film contains large quantities of red blood cell debris and the enlarged white blood cells. Your entire experiment, up to this point, has been to isolate that film at the bottom of the tube. 

5. Fixing the cells

• Add 5 mL of fixative solution to the centrifuge tube.
• With a pasteur pipette, mix the fixative and button of cells by drawing the mixture into the pasteur pipette and forcing it out again. Do this three of four times. Place this solution of cells and fixative into a refrigerator for 30 minutes. Make sure the test tube is covered with aluminum foil because of the smell. The 30 minutes in a refrigerator is a minimum; actually, it is possible to keep cells in the refrigerator overnight. During this time, practice dropping water on slides (instructions to follow).
• After refrigeration, centrifuge the tube for six minutes at 500 to 900 rpm.
• Remove the supernate and add another 6 mL of cold fixative and mix as you just did in the instructions above.
• Centrifuge the tube for six minutes at 500 - 900 rpm.
• Repeat the above two steps.
• Remove the supernate leaving about * mL of fluid at the bottom of the tube. It is this remaining material that you will drop on your slides in the next section. If you cannot see any material at the bottom of the test tube, do not despair; proceed as though there is visible material present. It is often very difficult to see.

6. Making the chromosome slides

• The slide must be exceptionally clean. Use new, factory pre-cleaned, frosted slide. The chromosome separation seems to work best if the slides are chilled in the freezer first.
• Lay five or six slides next to each other on paper toweling with no separation between them.
• Withdraw the entire contents of the centrifuge tube into a pasteur pipette. Be careful not to draw the fluid any farther than necessary into the pipette. The cells have a tendency to attach to the sides of the pipette.
• From a height of about 18 inches, drop two or three drops of fluid onto each side.
• Allow the slides to dry thoroughly. In fact, the best way to 'cure' the slides are to place them in the incubator (37oC) overnight.
• Stain the slide by immersion in fresh giemsa stain for 7 - 10 minutes.
• Remove the slides from the stain and rinse in distilled water until ALL the excess stain is removed.

Sources

Kaplan, B.J. (1978) Preparation of the normal karyotype (workbook). Chicago: American Society of Clinical Pathologists.
Macgregor, H.C. & Narley, J.M. (1983). Working with animal chromosome. New York: John Wiley & Sons

m2000 RealTime System- DNA/RNA Sample Preparation and Realtime PCR Complete Automation

Product Description

Automation is the foundation for the clinical molecular laboratory to report patient results with confidence. Abbott Molecular's m2000sp for sample preparation and the m2000rt for real-time amplification and detection are the cornerstrones for this process. m2000sp is modified to m24sp in some regions especially in Asia Pacific.

The Abbott m2000 RealTime System provides automation from bar-coded laboratory tube through patient result, creating an efficient workflow for your laboratory. This automation enables laboratorians to interface where their skills best contribute to results while minimizing errors and contamination.

Abbott Molecular is committed to molecular diagnostics. Whether through innovative assay design, expanding the mSystem menu, or new system software releases, we are continuously evolving.

m2000sp: Sample Extraction Automation

• Barcoded Laboratory Tubes - Reduces transcription error and provides positive sample ID
• Precision Pipetting - Eliminates manual mixing or manipulation
• Open Mode - Flexible protocol for various sample types and volumes
• Efficient Sample Extraction - Flexible throughput options of 24 to 96 samples

m2000rt: RealTime PCR

• Real-time PCR Amplification and Detection- 5 excitation, 5 emission filters
• maxRatio Data Analysis- Multiple validity checks for improved confidence in patient results
• Minimal Maintenance- Halogen bulb replacement
• Laboratory Defined Applications- Complete flexability in defining laboratory-based real-time PCR applications

m2000 System Software: Simplified Data Management

• Intuitive User Interface- Windows XPTM
• LIS Capability- Standardized interface with LIS
• Data Archiving - Calibrator, control, and patient data logs
• Automated Quality Checks and Calibration- Provides accurate results
• AbbottLink- Remote Instrument Monitoring

For more info, visit: Abbottmolecular.com

QIAcube- Fully Automated DNA and RNA Sample Preparation

QIAcube is a fully automated sample prep using QIAGEN spin-column kits. It uses advanced technology to process QIAGEN spin columns, enabling seamless integration of automated, low-throughput sample prep into the laboratory workflow. No change of purification chemistry is required, assuring fast startup and immediate results. All steps in the purification procedure are fully automated — up to 12 samples can be processed per run.

Performance

Highly pure plasmid DNA

The QIAcube together with the QIAprep Spin Miniprep Kit enables fully automated purification of up to 20 µg molecular biology grade plasmid DNA. The fully automated purification procedure — including lysis — provides yields of plasmid DNA comparable to the manual procedure (see figure “DNA yields comparable to the manual procedure”). Purified DNA is suitable for use in routine molecular biology applications, such as fluorescent sequencing, cloning, or transfection of robust cells.

High-quality genomic DNA

Using the QIAamp DNA Blood Kit, the QIAcube enables purification of reproducible yields of highly pure genomic DNA . DNA purified using the QIAcube performs well in sensitive PCR even when large amounts of eluate are used, demonstrating the high purity of the DNA.

Efficient purification of viral nucleic acids

The QIAcube together with the QIAamp MinElute Virus Spin Kit enables efficient purification of viral nucleic acids. To evaluate the risk of sample-to-sample carryover during and between runs, the QIAamp MinElute Virus Spin procedure was subjected to rigorous testing using an alternating checkerboard setup of negative and highly positive plasma samples (1 x 108 IU/ml of a typical DNA virus). All of the highly positive samples were detected. All negative samples, in the checkerboard runs and the all-negative runs were unresponsive (see table “No sample carryover detected”).

Pure, high performance RNA

Together with the QIAcube, the RNeasy Mini Kit enables efficient automated purification of total RNA from animal cells, demonstrated by consistent RNA yields. The quality of RNA purified using the QIAcube is comparable to the manual procedure.

High-quality proteins

The QIAcube and Ni-NTA Spin Kit provide reliable purification of highly pure 6xHis-tagged proteins. Purified proteins are suitable for many applications, including functional investigations, crystallization pre-studies for determination of three-dimensional structure, assays involving protein–protein and protein–DNA interactions, and immunization to produce antibodies.

Efficient cleanup of DNA fragments

The QIAcube makes DNA cleanup easier than ever. Proven QIAquick technology together with optimized protocols enables direct purification of double- or single-stranded DNA fragments, from 100 bp to 10 kb. Primers, nucleotides, enzymes, and other impurities are efficiently removed and recovery of DNA is comparable to the manual procedure.

Principle

The QIAcube enables continued use of well-established QIAGEN spin-column kits and eliminates the need for tedious manual steps. The innovative QIAcube controls integrated components including a centrifuge, heated shaker, pipetting system, and robotic gripper. This enables the QIAcube to fully automate more than 40 QIAGEN spin-column kits. 

The QIAcube is preinstalled with a variety of protocols for purification of RNA, genomic DNA, plasmid DNA, viral nucleic acids, and proteins, plus DNA and RNA cleanup. All standard protocols in the expanding range can also be downloaded free of charge. In addition, customized protocols tailored to meet your specific application demands can also be requested. 

Procedure

The QIAcube enables purification of highly pure nucleic acids or proteins using a simple lyse, bind, wash, elute procedure. Over 40 QIAGEN spin-column kits can be automated on the QIAcube.

Unparalleled ease of use

The QIAcube takes ease of use to a new level. No external PC is required, saving valuable laboratory space. An integrated touch screen simplifies protocol selection, and clear on-screen messages guide the user through worktable setup. For increased ease of use and high process safety, labware and accessories fit onto the worktable only in the correct orientation. In addition, a fully automated load check helps to ensure that samples, reagents, and labware are correctly loaded.

New dedicated kits for DNA and RNA preps

Dedicated QIAcube Kits further simplify automated spin preps on the QIAcube and increase convenience. Kits are currently available for purification of RNA, genomic DNA, and viral RNA. Rotor-adapters supplied with the kits are preloaded with spin columns and elution tubes, delivering greater convenience and time savings. Furthermore, ease of use is increased and user errors minimized. Waste is reduced as the content of the dedicated kits is tailored to purification on the QIAcube and tubes required for the manual procedure are not included.

Applications

The QIAcube is highly suitable for academic research laboratories as well as pharmaceutical, biotechnology, and biomedical research laboratories performing applications, such as:Sequencing/sequencing analysisGene expression analysisGenotypingProteomics

For more info, visit : qiagen.com

Understanding Polymerase Chain Reaction (PCR) Inhibitors

Understanding Polymerase Chain Reaction (PCR) inhibitors and how to avoid them is very essential for a scientist to have a good PCR product. Below is an article published by Pro Mega Corporation that focuses on approaches of identifying and overcoming PCR inhibitors.

Human Immunodeficiency Virus (HIV-1) RNA PCR Procedure (Qiagen Artus HIV PCR Kit)

Principle:

Pathogen detection by the PCR is based on the amplification of specific regions of the pathogen genome. The amplified product is detected via fluorescent dyes. These are usually linked to oligonucleotide probes which bind specifically to the amplified product. Monitoring the fluorescence intensities during the PCR run allows the detection and quantitation of the accumulating product without having to re-open the reaction tubes after the PCR run.

Hepatitis C Virus (HCV) PCR Test Procedure (Artus HCV RG PCR Kit)

Principle:

The pathogen detection by PCR is based on the amplification of specific regions of the pathogen genome. In real time PCR, the amplified product is detected via the fluorescent dyes. These are usually linked to the oligonucleotide probes which bind specifically to the amplified product.

Hepatitis B Virus (HBV) PCR Test Procedure (Artus HBV RG PCR Kit)

The artus HBV RG PCR Kit is a ready to use system for the detection of Hepatitis B Virus (HBV) DNA .

Principle:

The pathogen detection by PCR is based on the amplification of specific regions of the pathogen genome. In real time PCR, the amplified product is detected via the fluorescent dyes. These are usually linked to the oligonucleotide probes which bind specifically to the amplified product.

QIAmp DSP Virus Kit DNA/RNA Extraction Procedure

The QIAamp DSP Virus procedure combines the selective binding properties of a silica-based membrane with minimal elution volumes of 20 ul or 60 ul.

HIV and HIV (AIDS) Infection Overview

What is HIV?

The human immunodeficiency virus (HIV) is a member of the genus Lentivirus in the Retroviridae family, a large and diverse family of enveloped RNA viruses. Retroviruses are so called because their RNA genome is transcribed into linear double-stranded DNA by a characteristic enzyme known as reverse transcriptase (RT), a RNA-dependent DNA polymerase that reverses the classical flow of genetic information. The DNA subsequently enters the nucleus and integrates as a DNA provirus into the host cellular genome. The integrated retrovirus then is either transcriptionally active producing virions or remains in a silent or latent state. Lentiviruses consist of a diverse group of animal viruses with certain clinical and biological characteristics. The human counterpart, HIV, was discovered because of its association with the AIDS. This clinical condition is characterized by a marked reduction in the numbers of CD4þ T cells and a loss in immune function leading to the development of various opportunistic infections and cancer.

Viral RNA Mini Kit (QIAamp) RNA Extraction Procedure

Principle:
The QIAamp Viral RNA Mini Kit combines the selective binding properties of a silica-gel-based membrane with the speed of a microspin. The sample is first lysed under high denaturing conditions to inactivate RNases and to ensure isolation of intact viral RNA. Buffering conditions provide optimum binding of the RNA to the QIAamp membrane, and the sample is loaded onto the QIAamp mini spin column. The RNA binds to the membrane, and contaminants are efficiently washed away using two different was buffers. High quality RNA is eluted in a special RNAse-free buffer ready for direct use or safe storage.

DNA Blood Mini KIt (QIAamp) DNA Extraction Procedure

Principle: 

The QIAamp DNA Blood Mini Kit combines the selective binding properties of a silica-gel-based membrane with the speed of a microspin. Purification requires no phenol/chloroform extraction or alcohol precipitation, and involves very little handling. DNA is eluted in Buffer AE or water, ready for direct addition to PCR or other enzymatic reactions.

Polymerase Chain Reaction (PCR) Technique

The polymerase chain reaction (PCR) is a technique by which small samples of DNA can be quickly amplified, that is, increased to quantities that are large enough for analysis.

Hepatitis B Virus (HBV) PCR Test Procedure (Cobas TAqman By Roche)

Principle:
This is based on the two major processes: (1) manual specimen preparation to extract HBV DNA ;( 2) automated PCR amplification of target DNA using HBV specific complementary primers and detection of cleaved dual fluorescent dye-labeled oligonucleotide probes that permit quantitation of HBV target amplified product (amplicon) and HBV Quantitation Standard DNA, which is processed, amplified and detected simultaneously with the specimen.

Precautions:

• The use of sterile disposable pipets and D-nase free pipet tips is recommended. 
• Do not pool reagents from different lots or from different bottles of the same lot.
• Wear eye protection, laboratory coats and disposable gloves when handling any reagent. Avoid contact of these materials with the skin, eyes, or mucous membranes. If spills of these reagents occur, dilute with water before wiping dry.

Procedure:

A. Specimen Preparation:

Centrifuge whole blood for 20 minutes at 800-1,600 x g within two (2) hours & transfer the plasma into  sterile 2 ml polypropylene microcentrifuge tube.

B. Reagent Preparation :

• Pipette 20 ml of 100% Ethanol to Inhibitor Removal Buffer (IRB). Mix by inverting 5-10 times. Thisis enough for 48 tests. Transfer 5 ml into clean, sterile tube per each 12 samples.
• Pipette 80 ml of 100% Ethanol to Wash Buffer (WASH). Mix by inverting 5-10 times. This is enough for 48 tests. Transfer 20 ml into clean, sterile tube per each 12 samples
• Prewarm ELUTION BUFFER (EB) at 70⁰C in 2 ml microcen tube. 

          12 tests= 2.0 ml EB; 24 tests= 4.0 ml EB 

• Pipette 5 ml (12 tests)/or 10 ml (24 tests) isopropanol into a clean tube 
• Prepare 8.25 ml Inhibitor Removal Buffer (IRB)  + 5 ml 100% Ethanol for 12 tests/33 ml of Inhibitor Removal Buffer (IRB) + 20 ml 100% Ethanol
• Prepare LYSIS/BINDING WORKING SOLUTION as follows: 

Reagent	12 Test	24 Test
Lysis/Binding Buffer (12 tests)	7 ml	14 ml
Carrier RNA	140 ul	280 ul
HBV C	39 ul	78 ul
Proteinase K	1.4 ml	2.8  ml

C. DNA Extraction 

• Pipette 625 ul of prepared Lysis binding/carrier RNA QS/Proteinase Working Solution to each well of Lysis rack (transparent). 
• Pipette 500 ul of sample (plasma). Vortex for 10 sec to mix. 
• Incubate rack at 50 0C for 10 minutes 
• Centrifuge for 10-20 sec 
• Pipette 250 ul Isopropanol to each well. 
• Mix by inverting the rack 3 X, then vortex rack for 10 sec 
• Centrifuge for 10-20 seconds 
• Place filter tube rack (yellow) onto a waste container (white) & mark position 1 on the rack for orientation. 
• Transfer 750 ul of lysate to the wells of the filter tube rack. 
• Centrifuge for 2 min at 4600 g. 
• Transfer remaining lysate (750 ul) to the corresponding well of the filter tube rack. Discard lysis rack appropriately. Centrifuge for 2 min at 4600 g. 
• Remove filter tube rack from waste container & replace with a new waste. Discard waste container.
• Pipette 400 ul of IRB to each well. Push lids. 
• Centrifuge for 2 min at 4600 g. 
• Pipette 700 ul Wash Buffer, push lids, centrifuge for 2 mins at 4600 g. 
• Remove filter tube rack from waste container & replace with a new waste rack. 
• Pipette 700 ul of Wash buffer, push lids, centrifuge for 3 min at 4600 g. 
• Remove the filter tube rack from waste container & discard waste rack. 
• Place filter tube rack onto the elution rack (blue) and snap both racks.
• Pipette 75 ul of pre-warmed Elution Buffer onto the center of each filter. 
• Incubate elution rack at room temp for a minimum of 3 min after adding the Elution Buffer to last well. Centrifuge for 3 mins at 4600 g Remove the filter tube rack from the elution rack & discard filter rack.
• Place the cover rack (IIIB blue) onto the elution rack (IIIA blue). Close lids.
• The processed specimens and controls are now ready for PCR.

Note: Add the process specimens and controls to the Working Master Mix within 3 hours of preparation. Or store at 2-8⁰C for up to 24 hours, or frozen at -20⁰C for up to 1 week in Sterile 2 ml polypropylene microcentrifuge tube.

D. Amplification:

• Equilibrate HBV MMX and CTM Mn2 at ambient temperature for 30 mins. 
• Place K-carrier in a K-carrier holder. 
• Place new K-tubes in K-carrier without touching the sides of the K-tubes. 
• Prepare the working MMX as follows: For 12 tests, remove 660 ul of HBV MMX and place in a 2 ml tube. 
• Add 90 ul of CTM Mn2, cap the tube and mix well by inverting 10 times. 
• Protect the working MMX from light and use within 2 hours. 
• Pipette 50 ul of working MMX into each K-tube. 
• Add 50 ul of each processed specimen and control to the tubes containing the working MMX. Gently mix each specimen or control up and down three times without generating bubbles. Cap the tubes and ready to load in the thermal cycler.

Note:  If fewer than 24 tubes are to be run, positions 1, 2, 5, 20, 23, and 24 must be occupied to balance the K-carrier in the thermal cycler.

1	2	3	4	5
6	7	8	9	10
11	12		13	14
15	16	17	18	19
20	21	22	23	24

                 K- carrier format