|Products||Thermo Sequenase Dye Primer Manual Cycle Sequencing Kit from USB Corp.|
|Item||Thermo Sequenase Dye Primer Manual Cycle Sequencing Kit|
|Description|| The Thermo Sequenase Dye Primer Manual Cycle Sequencing Kit is designed to be used with fluorescent dye-labeled primers and high-resolution fluorescence scanners. Sequencing reactions involve a termination step followed by standard slab gel electrophoresis and scanning of the gel to detect fluorescently tagged sequencing products.|
The kit components have been optimized for sequencing directly from single bacterial colonies or plaques of M13 or lambda. No purification or pre-amplification is required. This kit permits rapid screening and sequencing of a large number of samples in a single working day. This kit can also be used for sequencing from 2 plasmids in yeast liquid culture. Isolation of yeast plasmids can be carried out using the USB Yeast Plasmid Isolation Kit
In all cases, the gel can be scanned without opening the glass plates, thus, allowing gel electrophoresis to be stopped at any point for analysis and then resumed if longer read lengths are needed.
Sequence from bacterial colonies, M13 or lambda plaques. Single yeast patches on plates or small liquid culture may be sequenced by using this kit combined with the Yeast Plasmid Isolation Kit, PN 79220 from USB.
Sequencing reactions involve use of fluorescent-labeled primers and a termination step. Note: The choice of fluorescent dyes depends on the scanner. For instruments equipped with a
532 nm laser (e.g. Hitachis FMBIO II or III Fluorescence Imaging Systems or Molecular Dynamics Typhoon), TAMRA (Rhodamine dye) or HEX (fluorescein dye) labeled primers work well. ROX and FAM labeled primers may also be used.
Thermo Sequenase DNA Polymerase/TAP Enzyme Mix
The enzyme is a mixture of Thermo Sequenase and thermostable inorganic pyrophosphatase (TAP) cloned from the thermophile Thermoplasma acidophilum. TAP prevents pyrophosphorolysis from occurring. Pyrophosphorolysis can result in faint bands, which can affect the accuracy of base calling.
Resolve Gel Compressions with 7-deaza-dGTP Nucleotide Mixes
When DNA is synthesized with 7-deaza-dGTP instead of dGTP, weaker secondary structures are formed, thus reducing compression artifacts in sequencing gels.
Specialized Formamide Loading Dye
A special formulation of the two tracking dyes does not obscure TAMRA or HEX primers. The faster migrating dye fluoresces at a wavelength greater than 649 nm. The slower migrating dye fluoresces at a wavelength greater than 600 nm. Thus, sequences will not be obscured if TAMRA or HEX labeled primers are used.
Direct Loading of Samples on the Gel
Alcohol precipitation of the sequencing reaction products is not required. Simply load the reaction onto a standard sequencing gel.
Components can also be used with radioactively labeled primers. However, for optimal results, a higher quality and quantity of DNA template is needed.
The formulation of Thermo Sequenase DNA Polymerase in this kit necessitates the use of a glycerol tolerant DNA sequencing gel to eliminate glycerol-induced distortion of bands at approximately 350 to 600 bases beyond the primer. Information on use is included in the kits protocol. If this region is beyond your reading range, Tris-Borate-EDTA Buffer (TBE) may still be used.
Thermo Sequenase with thermostable inorganic Pyrophosphatase
Reaction Buffer (concentrate)
Control DNA pUC19
Control Primer, TAMRA -40 Universal Forward Primer
ddG Termination Mix (7-deaza-dGTP)
ddA Termination Mix (7-deaza-dGTP)
ddT Termination Mix (7-deaza-dGTP)
ddC Termination Mix (7-deaza-dGTP)
Formamide Loading Dye
|Info|| USB Corp.|
26111 Miles Road
Cleveland Ohio 44128
Fax Number: 216.464.5075
Web Site: http://www.usbweb.com