DNA purification procedure utilizes spin minicolumns with membranes which efficiently and selectively bind nucleic acids. In the first step of the clean-up protocol CB Buffer is added to a DNA sample. It causes proteins to degrade and enables DNA binding to the column membrane while in the gel-out protocol DNA fragments is excised from an agarose gel and incubated in GB Buffer, which enables gel fragment solubilization and protein degradation. As an added convenience, the binding buffers contain a color 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. Purified DNA is eluted with the use 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.
CLEAN-UP: up to 200 μl of a DNA sample
GEL-OUT: agarose fragment of up to 300 mg containing DNA
Depending on DNA fragment length (in the range of 100 bp – 10 kb):
DNA FRAGMENT LENGTH
100 bp – 10 kb
DNA fragments in the range of 50–100 bp and 10–20 kb can also be purified as can genomic and plasmid DNA, however the efficiency will be decreased.
Approx. 40 μg DNA
5–10 min for clean-up procedure
16–20 min for gel-out procedure
A260/A280 ratio = 1.7 – 1.9
RECOMMENDATIONS AND IMPORTANT NOTES
Optimal volume of Elution Buffer used should be chosen in accordance with the amount of DNA in the sample and with final DNA concentration expected. The use of 30–100 μl of Elution Buffer is recommended. If high DNA concentration is desired the volume of elution may be reduced down to 20 μl. It should be noted that this may reduce efficiency. It is essential to apply Elution Buffer precisely onto 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, a second elution should be performed. For second elution, repeat steps 5 of the Isolation Protocol (section XI), placing the purification column in a new, sterile 1.5 ml Eppendorf tube.
Elution Buffer does not contain EDTA, which may interfere with some enzymatic reactions.
CB Buffer and GB Buffer contain 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 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.