PRINCIPLE

DNA purification procedure consists of four steps and utilizes spin minicolumns with membranes which efficiently and selectively bind nucleic acids. In the first step, biological material is lysed by Proteinase K in optimized GL Buffer. At this stage all the cellular membranes and proteins are  degraded. When it is necessary to remove RNA the use of RNase A is recommended. After the  addition of chaotropic salts, the lysate is applied to the purification minicolumns membrane and  the DNA is bound. The two-step washing stage effectively removes impurities and enzyme inhibitors. Purified DNA is eluted with the use of  either a low ionic strength buffer (Elution Buffer) or water (pH 7.0–9.0) and may be used directly in all downstream applications such as PCR, quantitive real-time PCR, pharmacogenic research, Southern blotting, single-nucleotide polymorphism (SNP), short tandem repeat (STR) genotyping, DNA sequencing, enzymatic restriction, 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
• physiological fluids (urine, PMR, peritoneal fluid): up to 5 ml
• cell culture: 10³–10⁷ cells
• broth or plate bacterial or yeast culture, frozen cell pellet
• buccal, nasal, pharyngeal, vaginal, blood and saliva swabs or semen
• fresh or frozen blood: up to 1 ml
• hair: 10–30 mg
• insects: 1–30 mg

EFFICIENCY

The typical efficiencies of DNA isolation from fresh biological material are given in section XIII.

BINDING CAPACITY

50 μg DNA

TIME REQUIRED

• approx. 12 minutes (lysis time not included)
• 10–60 minutes for sample preparation

DNA PURITY

A₂₆₀/A₂₈₀ ratio = 1.7 – 1.9

RECOMMENDATIONS AND IMPORTANT NOTES

DNA elution
An optimal volume of Elution Buffer used should be chosen in accordance with the quantity of sample material and final DNA concentration expected.

Tissue: The use of 100–200 μl Elution Buffer is recommended when extracting from 2–10 mg of tissue or <10⁴ cells. However, while extracting from 10–30 mg of tissue or 10⁴–10⁷ cells the volume of Elution Buffer should be increased to 200 μl.

Swab and Semen: The use of 50–100 μl Elution Buffer is recommended. The quantity of purified DNA depends on sample type and number of cells it contains; quality, however, on site’s features the swab was taken from and interindividual diversity. Usually the isolation efficiency from a single buccal swab is equal to 1–3 μg of DNA whereas, from 150 μl of semen to 2–7 μg. Bacteria and Yeast: The use of 50–100 μl Elution Buffer is recommended when extracting from no more than 10⁹ cells (bacteria) or 10⁸ cells (yeast). However, while extracting from a greater number of cells, the volume of elution buffer should be increased to 200 ul.

Blood: The use of 50–100 μl of Elution Buffer while extracting from 100–500 μl of blood is recommended. However, while extracting from 500–1000 μl of blood the volume of Elution Buffer should be increased to 200 ul. The quantity of purified DNA depends on the type of sample and the number of white blood cells it contains (patient’s age, his health condition, sample transport conditions, as well as storage time and method). Usually the isolation efficiency from 200 μl of blood sample from a healthy person is equal to 3–10 μg of DNA. A greater amount of DNA may be acquired from clinical samples containing increased number of white blood cells (3x 10⁶ – 1x 10⁷ cells/ml).