The EXTRACTME DNA CLEAN-UP & GEL-OUT KIT is designed for rapid and efficient purification of DNA fragments after enzymatic reactions and directly from agarose gels (standard and low-melting point agarose gels run in either a TAE or TBE buffer). It efficiently removes nucleases, enzyme inhibitors, detergents, restriction enzymes, polymerases, divalent ions, agarose, ethidium bromide and other contaminants. 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

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.

PRODUCT SPECIFICATIONS

SAMPLE MATERIAL

CLEAN-UP: up to 100ul of a DNA sample
GEL-OUT: agarose fragment of up to 300 mg containing DNA

YIELD

Depending on DNA fragment length (in the range of 100 bp – 10 kb):
CLEAN-UP: 90–99%
GEL-OUT: 70–95%

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 min for clean-up procedure
16–20 min for gel-out procedure

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 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 elution’s volume may be reduced down to 20 μl. It should be noted that this may reduce the efficiency of DNA retrieval. It is essential to apply Elution Buffer precisely onto the centre of the membrane. In order to maximize the DNA retrieval heat 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

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

pH monitoring

CB Buffer and GB Buffer contain an indicator, which enables pH monitoring. Yellow indicates that the solution’s pH is lower than 7.5, which guarantees optimal DNA binding with the membrane. When the pH is higher than 7.5, solution turns pink. It usually happens when the pH of a DNA sample considerably differs from the standard parameters of the DNA treatment operations (pH > 9.0, when the running buffer for electrophoresis has been used several times or was incorrectly prepared). In such cases, 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.