PAXgene Blood DNA Kit
For purification of up to 500 µg genomic DNA from whole blood
- Efficient purification of high-quality, high-molecular weight DNA
- Optimized DNA purification from specimens collected in PAXgene Blood DNA Tubes
- Rapid procedure in a single processing tube
- Standardized and easy workflow
|Technology||Lysis, digestion, and precipitation|
|Sample input volume||Content of one PAXgene Blood DNA Tube|
|Elution volume||1 ml|
|Sample type||8.5 ml whole blood, drawn into a PAXgene Blood DNA Tube|
|Time per run||60 min/8 samples|
|Typical DNA yield||150–500 µg|
|DNA purity||1.7–1.9 (A260/A280)|
|DNA size||50–200 kb|
The PAXgene Blood DNA Kit is intended for the purification of DNA from whole blood samples. It is intended to be used as part of the PAXgene Blood DNA System, which additionally includes the PAXgene Blood DNA Tube for the collection and storage of whole blood.
For research use only. Not for use in diagnostic procedures. No claim or representation is intended to provide information for the diagnosis, prevention, or treatment of a disease. The performance characteristics of this product have not been fully established.
Blood samples (8.5 ml) are collected in PAXgene Blood DNA Tubes, where they may be stored or transported. For DNA isolation, the blood is transferred to processing tubes (supplied already filled with cell lysis buffer), and the solution is mixed to lyse red and white blood cells. Cell nuclei and mitochondria are pelleted by centrifugation, washed, and resuspended in digestion buffer. Protein contaminants are removed by incubation with a protease. DNA is precipitated in isopropanol, washed in 70% ethanol, dried, and resuspended in resuspension buffer.
Multiplex PCR of fragments from the mitochondrial genes tRNAlys/ATPase (0.92 kb), tRNAleu(UUR) (0.63 kb), and ND4 (0.29 kb), using 250 ng DNA from 8 donors as starting material.
Genomic DNA isolated from 8 blood donors using the PAXgene Blood DNA System. [A] Agarose gel analysis; [B] pulsed-field gel electrophoresis for enhanced separation of high-molecular-weight genomic DNA. M: markers.
Multiplex PCR from gene fragments CD19 (955 bb), CD59 (845 bp), CD40 (756 bp), CD14 (662 bp), AGTR (523 bp), cKit (414 bp), B29 E4 (310 bp) and PRP (222 bp), using 250 ng DNA isolated from 8 blood donors using the PAXgene Blood DNA System as starting material. M: markers.
Amplification of a 15 kb fragment of the human coagulation factor IX gene, using 250 ng DNA isolated from 8 blood donors using the PAXgene Blood DNA System as starting material. M: markers.
DNA from whole blood samples collected in PAXgene Blood DNA Tubes purified using the PAXgene Blood DNA Kit has an A260/A280 ratio of 1.7–1.9. Fragments range between 20–200 kb in size, with an average length of 50–150 kb (see High quality and high molecular weight of genomic DNA). Average DNA yields are 150–500 µg, depending on the number of nucleated cells present in the blood sample.
Applications such as pharmacogenomic studies, gene expression studies, and SNP genotyping yield clinically important data and can require large amounts of purified DNA from individual subjects. Use of standardized methods for sample collection and nucleic acid isolation to produce high-quality DNA for such studies is becoming increasingly important for multi-center clinical trials and basic research. The purification process for the PAXgene Blood DNA Kit is performed in a single processing tube to minimize the risk of sample mix-up and cross-contamination. Together with the PAXgene Blood DNA Tube, which standardizes the collection and storage of whole blood at various temperatures, these two components of the PAXgene Blood DNA System comprise a simplified, integrated and standardized preanalytic workflow for blood samples used in DNA studies.
The procedure to purify DNA using the PAXgene Blood DNA Kit is shown in the flowchart PAXgene Blood DNA procedure. Briefly, blood samples collected in PAXgene Blood DNA Tubes are transferred to processing tubes (supplied already filled with cell lysis buffer), and the solution is mixed to lyse red and white blood cells. Cell nuclei and mitochondria are pelleted by centrifugation, washed, and resuspended in digestion buffer. Protein contaminants are removed by incubation with a protease. DNA is precipitated in isopropanol, washed in 70% ethanol, dried, and resuspended in resuspension buffer.
The resulting high-quality DNA can be used for demanding downstream applications, including:
- PCR, multiplex, long-range, and quantitative, real-time PCR
- Southern blotting
- SNP genotyping
|Reducing Variability and Improving Workflow in the Collecting, Transporting, and Processing of Blood Samples for Genomic DNA Purification Using the PAXgene Blood DNA System; Groelz et al., ASHG, 2004||917 KB|
DNA Isolation and Processing
1. How should I thaw frozen PAXgene Blood DNA Tubes?
Thaw the PAXgene Blood DNA Tubes in a wire rack at ambient temperature (18–25ºC) for approximately two hours or at up to 37°C in a water bath for approximately 15 minutes. Carefully invert the thawed PAXgene Blood DNA Tubes 10 times.
2. What is the expected yield from 8.5 ml of blood?
Yield of genomic DNA will vary depending on the cell count from the donor blood sample but average range is from 150 µg to 500 µg.
3. What is the expected purity and size of DNA?
The typical A260/A280 ratio is from 1.7 to 1.9. The DNA molecules are up to 200 kb in size, with fragments of 50–150 kb predominating.
4. Can mitochondrial DNA be isolated with the PAXgene Blood DNA System?
Yes, mitochondrial and genomic DNA are both isolated.
5. Does the PAXgene Blood DNA System contain RNA contamination?
No. DNA purified with the PAXgene Blood DNA system is free of detectable RNA contamination. In contrast to commercial salting out methods, additional RNase digestion steps are not necessary.
6. Can smaller volumes of blood be processed from the PAXgene Blood DNA Tube?
No. The amount of Buffer BG1 (lysis buffer) prefilled in the processing tubes, and all the other buffer volumes are optimized for use with blood from one PAXgene Blood DNA Tube.
7. What does the resuspension buffer, Buffer BG4, contain?
Buffer BG4 consists of 10 mM Tris·Cl, pH 8.5.
8. How should DNA purified using the PAXgene Blood DNA System be stored?
DNA purified using the PAXgene Blood DNA System should be dissolved in Buffer BG4 and stored at 2–8°C. For long-term storage, DNA can be stored at –20°C but multiple freezing and thawing cycles should be avoided.
9. Is it possible to interrupt the protocol?
Yes. After dissolving the nuclei pellet in digestion buffer, Buffer BG3 (step 7), samples can be stored up to 7 days at 2–8°C or for one day at room temperature (18–25°C). The protocol can also be interrupted after adding isopropanol (step 10).
10. Can I use the PAXgene Blood DNA Tube to collect blood from animals?
The system is designed for use only with human blood samples.
11. Can I add bleach to the sample preparation waste?
Do not add bleach or acidic solutions directly to the sample preparation waste. CAUTION: the sample preparation waste contains guanidinium hydrochloride from Buffer BG3, which can form highly reactive compounds when combined with bleach. For more information please consult the appropriate material safety data sheets (MSDS).
12. What downstream applications have been tested with DNA purified using the PAXgene Blood DNA System?
So far, we have shown that DNA purified using the PAXgene Blood DNA System can be used in PCR, including long range and multiplex PCR, restriction digestion and Southern analysis, as well as in various SNP detection procedures including,TaqMan, LightCycler and Masscode technologies.
|Product||Catalog Nr.||Price (USD)|
PAXgene Blood DNA Kit (25)Processing tubes and buffers for 25 preparations. To be used in conjunction with PAXgene Blood DNA Tubes.