clonewell

Frequently Asked Questions

Q - How much DNA can I load in the 20–25 µL sample?

A - Load 500–700 ng of total sample; load 50–200 ng DNA per band, though 20–400 ng per band is acceptable. Higher amounts may require two fractions.


Q - What buffer is the DNA eluted into?

A - Distilled water. The Collection Wells are filled with distilled water at the beginning of the run and then refilled as the band of interest reaches the Reference Line. This allows elution of the DNA band into distilled water with minimal salts from the gel.


Q - What if I missed the band of interest in the collection well?

A- If the DNA of interest runs past the collection well, stop the run by pressing the “Go” button. Run the gel on Program 5, REVERSE E-Gel until the DNA re-enters the collection well.


Q - What volume do you recover the DNA into?

A - DNA bands are recovered in 20–25 µL of distilled water.


Q - What can I do to get a higher DNA concentration of the retrieved DNA?

A - The recommended volume for optimal yield is 20–25 µL. However, when higher concentration of DNA is needed and yield is not a concern, the band can be retrieved in 10–20 µL of distilled water.


Q - How much salt is in the purified DNA sample and how will it affect ligation reactions?

A - If the sample is retrieved in distilled water, the concentration of salts in the isolated sample is minimal and there is no need to desalt the sample prior to downstream applications such as cloning.


Q - Can I use the Ethidium Bromide Double Comb E-Gel® gel instead of the CloneWell™ gel?

A- No, using an Ethidium Bromide gel will require a UV light source, which could damage the DNA and expose the user to UV light. While the ion reservoir of the Ethidium Bromide Double Comb E-Gel® gel supports short runs (around 15 minutes) the ion reservoir of the CloneWell™ gel is increased to allow a longer run so that the larger bands can reach the collection well.


Q - What methods are compatible with the CloneWell™ gel?

A - DNA extracted from CloneWell™ gels is compatible with common cloning methods such as restriction cloning, TOPO® cloning, and Gateway® cloning.


Q - Has anyone tried to sequence the DNA after it was run on a CloneWell™ gel?

A - Yes, a Life Technologies R&D scientist has successfully sequenced a 350 bp fragment that was extracted using a CloneWell™ gel. The results were good and were comparable to sequence obtained from a PCR product extracted from a conventional ethidium bromide gel. You may also want to review a publication by Gibson, et al. (2010) “Band-cutting no more: A method for the isolation and purification of target PCR bands from multiplex PCR products using new technology” Molecular Phylogenetics and Evolution 56:1126–1128. Life Technologies Demonstrated Protocols have been successfully demonstrated by Life Technologies Research and Development but not formally validated. There are no technical specifications for Life Technologies demonstrated protocols. Users assume all risk when using these protocols, and recognize that support for Life Technologies demonstrated protocols occurs through community discussion. All customers are encouraged to discuss and contribute via the Ion community website (http://ioncommunity.lifetechnologies.com/welcome).

For Research Use Only. Not for use in diagnostic procedures. The trademarks mentioned herein are the property of Life Technologies and/or its affiliate(s) or their respective owners.