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High Pure PCR Template Preparation Kit
Low to medium throughput genomic DNA isolation
Application
Use this High Pure kit to purify nucleic acids from a wide variety of sample materials (e.g ., whole blood, cells in culture, tissue). The isolated nucleic acids can be used for:
•Long template PCR
•Real-time, quantitative PCR
•SNP detection
•Southern blotting
•Cloning
Benefits
•Purifies nucleic acids from many sources
•Reduces handling steps, minimizing the loss and fragmentation of DNA
•Can recover high molecular weight DNA (30 -50 kb) that is suitable for long template PCR
•Allows quantitative removal of contaminants, to improve the reliability and reproducibility of downstream procedures (e.g., real-time, quantitative PCR)
•Can save time by preparing multiple PCR templates simultaneously
Product Description
Sample Material
•200 – 300 μl mammalian whole blood
•200 μl buffy coat
•104 – 106 cultured mammalian cells
•25 – 50 mg mammalian solid tissue
•0.2 – 0.5 cm mouse tail (25 - 50 mg)
•108 yeast cells
•109 bacterial cells (gram positive or gram negative)
•Paraffin-embedded, fixed tissue sections
Background Information
Nucleic acids (NA) bind to the surface of glass fiber fleece in the presence of chaotropic salt. This allows the High Pure filter tube to specifically immobilize nucleic acids (both DNA and RNA) while they are freed of contaminants.
Contents
1.Tissue Lysis Buffer, 20 ml
2.Binding Buffer, 20 ml
3.Proteinase K, recombinant PCR grade
4.Inhibitor Removal Buffer, 33 ml
5.Washing Buffer, 20 ml
6.Elution Buffer, 40 ml
7.High Pure Filter Tubes, two bags with 50 polypropylene tubes with two layers of glass fiber fleece, for use of up to 700 μl sample volume
8.Collection Tubes, eight bags with 50 polypropylene tubes (2 ml)
Principle
Cells are lysed during a short incubation with Proteinase K in the presence of a chaotropic salt (guanidine-HCl), which immediately inactivates all nucleases. Cellular nucleic acids (NA) bind selectively to special glass fibers pre-packed in the High Pure Purification Filter Tube. Bound NA is purified in a series of rapid "wash-and-spin" steps to remove contaminating cellular components. A special Inhibitor Removal Buffer has been included which allows even the application of heparinized sample material with - 100 U/ml of Heparin. Finally, low salt elution releases the NA from the glass fiber. This simple method eliminates the need for organic solvent extractions and DNA precipitation, allowing for rapid purification of many samples simultaneously.
Test Principle
•Blood, cells or tissue are lysed by incubation with a special Lysis Buffer and Proteinase K.
•Nucleic acids are bound to the glass fibers pre-packed in the High Pure Filter Tube.
•Bound nucleic acids are washed with a special Inhibitor Removal Buffer to get rid of PCR inhibitory contaminants.
•Washing of bound nucleic acids, purification from salts, proteins and other cellular impurities.
•Purified Nucleic Acids are recovered using the Elution Buffer.
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