The DNA tethering kit is a highly efficient system to tether a DNA sequence of interest (SOI) in optical tweezers and study dynamics of DNA-binding proteins. For tethering nucleosome arrays with this kit, have a look at our nucleosome array tethering protocol.
Using the kit, the SOI can be ligated to two biotinylated DNA handles of 6,298 bp length each. The default kit comes with one ATTO647N-labeled handle and one ATTO488-labeled handle, in case you need variations of the handles without fluorophores, please contact store@lumicks.com. Both handles have a single– stranded overhang of 4 nucleotides for the DNA Ligase reaction. In the protocol, a method to introduce matching overhangs in the SOI is described using a digestion with a TypeIIS restriction enzyme of choice among BsmBI, BbsI or BsaI. The restriction sites can be introduced in the SOI either by PCR or by using the pUC-LUMICKS plasmid included in the kit.
Both handles include a fluorophore (one handle with ATTO 647N and the other handle with ATTO 488) which will flank the SOI and can be used to position the SOI on the focal plane before incubation with fluorescent proteins. This enables setting up optimal fluorescence imaging conditions before starting a DNA-protein interaction measurement, which allows capturing even the first interactions. Because the distance between the fluorophores is known, the fluorophores also serve as ruler on the tethered DNA to precisely determine the position on the DNA sequence of the fluorescent protein interacting with the SOI.
In addition to the DNA handles and the pUC-LUMICKS plasmid, the kit also contains a DNA Ligase enzyme, 10x DNA Ligase buffer, beads and a short DNA construct (3,065 bp) with overhangs that can be used as a quality control for the labeling and tethering procedures.
Our DNA tethering kit relieves you from costs and time that are typically required for personnel training, troubleshooting, experiment optimization, and custom application development.
The kit enables 4 DNA Ligase reactions. Each DNA ligase reaction can be used for at least 5 experimental sessions on the C-Trap. Please note that the current price of the product is an temporary, introduction offer.
In case you are interested in variations of the handles without fluorophores, please contact store@lumicks.com.
Estimated 1-4 weeks delivery time.
