PRINCIPLE

DNA purification procedure consists of four steps and utilizes spin DNA Purification Columns with membranes which efficiently and selectively bind nucleic acids. In the first isolation step, tissue is homogenized in order to disintegrate intercellular bonds (epithelial tissue) and fragmentize high-molecular proteins (muscle or connective tissue). Then homogenate is lysed by Proteinase K in optimized TL Buffer. At this stage, all the cellular membranes and proteins are degraded. When a metabolically active tissue is used for isolation, RNA is removed by RNase A. Homogenate is separated from undigested tissue remains by centrifugation and combined with chaotropic salts. Mixture is then applied to DNA Purification Column membrane and DNA is bound. A two-step washing stage effectively removes impurities and enzyme inhibitors. Purified DNA is eluted using a low ionic strength buffer or water (pH of 7.0–9.0) and can be used directly in all downstream applications such as qPCR, Southern blotting, DNA sequencing, enzymatic restriction, DNA ligation and so forth, or stored until ready to use.

PRODUCT SPECIFICATIONS

SAMPLE MATERIAL

• fresh or frozen solid tissue: 1–30 mg
• formalin-preserved tissue: 1–30 mg
• paraffin-embedded tissue: 1–30 mg
• cell culture: 10₃–10₇ cells
• physiological fluids (urine, PMR, peritoneal fluid): 1–5 ml
• hair: 10–30 mg
• insects: 1–30 mg
• rodent tail: up to 30 mg

BINDING CAPACITY

Approx. 50 μg DNA

TIME REQUIRED

• approx. 12 minutes (lysis time not included)
•  30–40 minutes for mechanical homogenization
• 1–16 hours for periodical shaking by vortexing

DNA PURITY

A²⁶⁰/A²⁸⁰ ratio = 1.7 – 1.9

RECOMMENDATIONS AND IMPORTANT NOTES

RNA contamination
Most fresh or frozen tissue contains more RNA than DNA, especially metabolically active tissues like glands, nerve tissue and epithelium. RNA may interfere with some enzymatic reactions, but does not inhibit PCR. If an RNA-free DNA sample is desired, add 4 μl of RNase A solution and incubate at 37°C for 5 minutes (step 3 of the Isolation Protocol, section XI).