PRINCIPLE

DNA purification procedure utilizes spin minicolumns with membranes which efficiently and selectively bind nucleic acids. First, CB Buffer is added to DNA sample. It causes proteins to degrade and enables DNA to bind with the column’s membrane. The binding buffer contains a color indicator, that facilitates an 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 using 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, qPCR, DNA sequencing, enzymatic restriction, DNA ligation, etc. or stored until ready to use.

PRODUCT SPECIFICATIONS

SAMPLE MATERIAL

up to 200 μl of DNA sample

YIELD

60-99% – depending on DNA fragment length

DNA FRAGMENT LENGTH

50 bp – ~20 kbp
genomic and plasmid DNA, however the efficiency will be decreased

BINDING CAPACITY

approx. 40 μg DNA

TIME REQUIRED

10 min for 6 PCR purifications

DNA PURITY

A_260/280 ratio = 1.7-1.9

RECOMMENDATIONS AND IMPORTANT NOTES

DNA elution

The optimal volume of Elution Buffer used should be chosen in accordance with the amount of DNA in a sample and to final DNA concentration expected. The use of 15-30 μl of Elution Buffer is recommended. It is essential to apply Elution Buffer precisely onto the centre of the membrane. In order to maximize DNA recovery, the following modifications should be applied:

• Heat Elution Buffer up to 70°C, apply onto the column and incubate at room temperature for 5 min.
• Carry out 2 or 3 elution steps with 15-30 μl Elution Buffer. To assure complete DNA recovery from the membrane, use 200 μl Elution Buffer.

Elution Buffer

Elution Buffer does not contain EDTA, which may interfere with some enzymatic reactions.

pH monitoring

CB 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.