Genomic DNA isolation, Lysis Buffer B3, Macherey-Nagel™

Description

Principles/procedure for purification of genomic DNA from whole blood
NucleoSpin™ Blood kits are designed for rapid purification of genomic DNA from whole blood, serum, plasma and body fluids. Animal or human blood samples, fresh or frozen material, can be processed conveniently. The NucleoSpin™ Blood method is based on a specially treated silica membrane with a high binding capacity for nucleic acids. The obtained DNA can be used directly for PCR* (e.g., HLA typing), Southern blotting, or any kind of enzymatic reaction. The kit allows isolation of 4μg to 6μg of pure genomic DNA from 200μL whole blood with an A_260/280 ratio between 1.60 and 1.90 and a typical concentration of 40ng/μL to 60ng/μL. Due to the high binding capacity of the NucleoSpin™ Blood membrane, yields of up to 60μg are possible (from starting materials like buffy coat or lymphocyte-enriched samples). With the NucleoSpin™ Blood method, genomic DNA is prepared from whole blood. Lysis is achieved by incubation of whole blood in a solution containing large amounts of chaotropic ions in the presence of proteinase K at 70°C. Appropriate conditions for binding of DNA to the silica membrane of the NucleoSpin™ Blood columns are created by addition of ethanol to the lysate. The binding process is reversible and specific for nucleic acids. Contaminations are removed by washing with two different buffers. Pure genomic DNA is finally eluted under low ionic strength conditions in a slightly alkaline elution buffer. Time for preparation of genomic DNA from blood with NucleoSpin™ Blood is less than 30 minutes.
Principles/procedure for purification of viral DNA from blood and biological fluids
NucleoSpin™ Blood kits are highly sensitive for the preparation of viral nucleic acids from serum, plasma or blood. The isolation of viral DNA – especially from Hepatitis B (HBV) – from serum and plasma has become more important with the growing demands of clinical diagnostics. The extraction of HBV DNA is demanding due to the special genome organisation of the Hepatitis B virus. A terminal protein, covalently bound to one of the HBV DNA strands, has to be removed prior to binding of the nucleic acid to the silica membrane. The NucleoSpin™ Blood method includes a proteinase K step for digestion of proteins. Subsequently, the viral DNA is bound to the silica membrane and washed twice with two different buffers ensuring removal of contaminations. Elution can be performed with water or low-salt buffer. Kit components: NucleoSpin™ Blood columns, 2mL collecting tubes, buffers, proteinase K.

• Preparation time: 30min/prep
• Silica membrane technology
• Format: mini spin columns
• Complete removal of PCR* inhibitors
• Sample size: up to 200μL/ 5x10⁶ cells
• Typical yield: 4μg to 6μg DNA
• Elution volume: 100μL
• Binding capacity: ≤60μg
• No use of organic solvents
• Also suitable for isolation of viral DNA: HBV,cmV, HPV and EBV For extraction from: Whole blood (human or animal blood); Whole blood treated with citrate, EDTA, heparin CPDA; Serum, plasma, buffy coat, platelets, body fluids (e.g., amniotic fluid); Up to 10⁷ lymphocytes; Cultured cells. Viral nucleic acid isolation - HBV;cmV; HPV; EBV.