The EXTRACTME DNA CLEAN-UP KIT is designed for the rapid and efficient purification of DNA fragments after enzymatic reactions. It efficiently removes nucleases, enzyme inhibitors, detergents, restriction enzymes, polymerases, divalent ions, salts etc. The purified DNA can be used in common downstream applications. The kit enables the purification of DNA fragments from 50 bp to 30 kb, as well as plasmid and genomic DNA. However purification of fragments smaller than 100 bp and larger than 10 kb will result in decreased recovery rates. The purification protocol and buffer formulations were optimized for high yields and purity of DNA. The product is intended for research use only.

PRINCIPLE

The DNA purification procedure utilizes spin minicolumns with membranes which efficiently and selectively bind nucleic acids. In the first step the CB Buffer is added to a DNA sample. It causes proteins to degrade and enables DNA binding to the column membrane. As an added convenience, the 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

up to 100 μl of a DNA sample

YIELD

90-99%, depending on DNA fragment length (in the range of 100 bp – 10 kb)

DNA FRAGMENT LENGTH

100 bp – 10 kb
DNA fragments in the 50-100 bp and 10-30 kbp range can also be purified, as can genomic and plasmid DNA, however the efficiency will be decreased.

BINDING CAPACITY

approx. 25 μg DNA

TIME REQUIRED

5-10 minutes

DNA PURITY

A₂₆₀/A₂₈₀ ratio = 1.7-1.9

RECOMMENDATIONS AND IMPORTANT NOTES

DNA elution

The optimal volume of the elution buffer used should be chosen in line with the amount of DNA in the sample and to the final DNA concentration expected. The use of 30-100 μl of the Elution Buffer is recommended. If a high DNA concentration is desired, the elution volume may be reduced down to
20 μl. It should be noted that this may reduce the efficiency of the DNA retrieval. It is essential to apply the elution buffer precisely onto the centre of the membrane. In order to maximize the DNA retrieval heat the Elution Buffer to 80 ̊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 10-13 of the Isolation Protocol (section XI), placing the purification column in a new, sterile 1.5 ml Eppendorf tube.

Elution Buffer

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

pH monitoring

The CB Buffer contains an indicator, which enables pH monitoring. Yellow indicates that the pH of the solution is lower than 7.5, which guarantees optimal DNA binding to the membrane. When the pH is higher than 7.5, solution will turn pink. It may happen on the occasion, when the pH of a DNA sample considerably differs from the standard parameters of the DNA treatment operations (pH>9.0). In this case, it is essential to add 10 μl 3 M sodium acetate (pH 5.2). It will lower the pH, enabling the solution to bind efficiently to the minicolumn membrane.