Roche's fast and high-yield new product: MagNA Pure 96 DNA and viral nucleic acid kit
Foreword
MagNA Pure 96 is a new kit that has recently entered the market: it is a fully automated, ultra-high-speed system that can be used to separate high-quality nucleic acids from various sample types at high speeds, and can be completed in less than 1 hour. Up to 96 samples are automatically purified, and the processed sample volume is up to 1 ml. The system uses an optimized kit equipped with pre-installed ready-to-use reagents and is equipped with corresponding automatic separation methods. The configuration of this kit is based on the well-established magnetic bead technology, which has been successfully used in different MagNA Pure systems for many years. The MagNA Pure 96 kit consists of reagent trays with different pre-loaded sealed buffer containers and special bottles containing magnetic powder. All kits available have different specifications for small volume (SV) and large volume (LV), which can be determined according to the amount of sample to be processed. This article describes the characteristics and performance of two MagNA Pure 96 DNA and viral nucleic acid kits and their methods.
Materials and Method
The new kit and method were developed and tested using human EDTA plasma, citrate plasma, serum and whole blood, and cultured cells (K562 and HeLa) as sample materials.
Whole blood can be used for genomic DNA isolation without pretreatment. The cultured cell pellets must be suspended in PBS before use.
During the isolation of viral nucleic acids, different amounts of DNA viruses (cytomegalovirus, Epstein-Barr virus, parvovirus B19) and RNA viruses (hepatitis A virus, H1N1 influenza virus) are added to the sample before use. Then pipette the sample into the MagNA Pure 96 process cartridge on the MagNA Pure 96 instrument. All samples are tested with at least 8-fold replication. The elution volume of purified nucleic acid is set to 50 μl, 100 μl, or 200 μl according to experimental needs. The eluate was analyzed on the LightCycler® 480 using absorbance (OD) measurement, agarose gel electrophoresis, and / or parameter-specific quantitative real-time PCR and RT-PCR analysis. 15 μl of each mixed mother liquor and 5 μl of each eluate were used to establish an amplification reaction, and 45 cycles were run.
Results and discussion
General characteristics The composition of the kit: pre-loaded reagent containers arranged in the reagent tray for easy loading; separate bottles containing magnetic beads (MGP) suspension (Figure 1).
Reagent trays and bottles are placed in their respective positions in the instrument drawer before operation. After use, the container can be easily resealed and stored for subsequent operation when necessary. The MGP bottle has a special bottle cap, which can be automatically resealed after the needle of the instrument passes through. All components are stable at room temperature.
MagNA Pure 96 DNA and viral nucleic acid small capacity specifications are used to isolate genomic DNA from 200 μl whole blood, or from 5 × 105 cultured cells. It can also isolate viral DNA or RNA from 200 μl of plasma, serum or whole blood. The kit includes 3 pre-loaded reagent containers, each of which can carry out up to 192 reactions (that is, 576 purifications per kit).
MagNA Pure 96 DNA and viral nucleic acid kits are available in large-capacity formats for isolating genomic DNA from 500 μl or 1000 μl whole blood or from 106 cultured cells. It can isolate viral DNA or RNA from 500μl plasma, serum or whole blood, or from 1000μl plasma or serum. The kit includes 3 pre-loaded reagent containers, each of which can carry out up to 96 reactions (500 μl) or 48 reactions (1000 μl) (that is, each kit performs 288 or 144 purifications, respectively).
If needed, the kit can be used for different optimization methods, including an external dissolution method, which allows virus inactivation outside of the MagNA Pure 96 instrument. For all methods, nucleic acids can be easily eluted from samples of different volumes, and different volumes of samples can be selected by the user.
Genomic DNA
The isolation and subsequent analysis of genomic DNA showed high repeatability, yield, integrity and purity. According to agarose gel analysis, the average DNA fragment was significantly larger than 20 kb (Figure 2).
Table 1 summarizes typical DNA yields and purity. Please note that the yield depends significantly on the number of blood cells, so the change may be up to 2 times.
As shown in the dilution experiment before PCR performed in the LightCycler® 480 instrument (data not shown), the DNA was also proven to be free of PCR inhibitors.
The scalability of the program was tested with different amounts of blood and samples. The results are shown in Figure 3.
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