PRINCIPLE

The DNA purification procedure utilizes spin minicolumns with membranes which efficiently and selectively bind nucleic acids. In the first step, the DNA fragment is excised from an agarose gel and incubated in GB Buffer, which enables gel fragment solubilization and protein degradation. As an added convenience, binding buffer contains a colour indicator, which facilitates easy monitoring of the solution’s pH for optimal DNA binding. The two-step washing stage efficiently removes impurities and enzyme inhibitors. The purified DNA is eluted using either a low ionic strength buffer (Elution Buffer) or water (pH 7.0–9.0) and can be used directly in all downstream applications such as PCR, qPCR, Southern blotting, DNA sequencing, enzymatic restriction, ligation and so forth or stored until ready to use.

PRODUCT SPECIFICATIONS

SAMPLE MATERIAL

Agarose fragment of up to 300 mg containing DNA

YIELD

70-95%, depending on DNA fragment length (in the range of 100 bp – 10 kb)

DNA FRAGMENT LENGTH

50 bp – 20 kb
Genomic and plasmid DNA, however, the efficiency will be decreased.

BINDING CAPACITY

Approx. 40 μg DNA

TIME REQUIRED

16-20 minutes

DNA PURITY

A₂₆₀/A₂₈₀ ratio = 1.7 – 1.9

RECOMMENDATIONS AND IMPORTANT NOTES

DNA elution

Optimal volume of Elution Buffer used should be chosen in line with the type of DNA in a sample and final DNA concentration expected. The use of 50–100 μl of Elution Buffer is recommended. If a high DNA concentration is desired, elution volume may be reduced to 20 μl. It should be noted that this may reduce efficiency. It is essential to apply Elution Buffer precisely to the centre of the membrane. In order to maximize DNA retrieval, heat Elution Buffer to 70°C and incubate it on the membrane for 10 minutes. If full DNA retrieval is required 200 μl Elution Buffer should be used. However it will result in DNA dilution. Second elution can be also be performed. For second elution, repeat steps 11–14 of the Isolation Protocol (section XI), placing purification column in a new, sterile 1.5 ml Eppendorf tube. Elution Buffer does not contain EDTA, which may interfere with some enzymatic reactions.

pH monitoring

GB Buffer contains an indicator, which enables pH monitoring. Yellow indicates that the solution’s pH is lower than 7.0 and guarantees an optimal DNA binding with the membrane. When the pH is higher than 7.0, solution turns pink. It usually happens when the pH of DNA sample considerably differs from the standard parameters of DNA treatment operations (pH>9.0). In this case, it is essential to add 10 μl of 3 M sodium acetate (pH 5.2). It will lower the pH, enabling the solution to bind efficiently with the minicolumn membrane.

Loading Buffer

Loading Buffer is provided for analysis of purified DNA samples with the use of gel electrophoresis. Loading Buffer contains 3 dyes (bromophenol blue, xylene cyanol and orange G). Loading Buffer is concentrated by a factor of six, thus, in order to obtain the most satisfying results mix 2 μl of Loading Buffer with 10 μl of purified DNA.