DNA Sequencing - Sample Submission Guidelines
All DNA templates and primers, should be supplied in either molecular biology grade water, or 10 mM Tris-HCl. Please avoid buffers containing EDTA (ie. TE Buffer). All tubes should be clearly labeled, sealed securely and must be accompanied by a signed requisition form. Results may be delayed if information is lacking from the order form.
Sequencing results will be provided will be provided by email in both .txt and .ab1 format.
Samples for capillary electrophoresis are loaded into the capillary via electrokinetic injection (EI) . During EI the negatively charged DNA is attracted to the positively charged capillary tip and migrates through the liquid polymer at a predefined rate. With EI, sample conductivity determines what is injected into the capillary, salts and other charged molecules compete with and may reduce the number of DNA molecules which are injected into the capillary resulting in loss of signal. In addition, proteins, RNAs, detergents and unlabeled DNA templates can cause fouling of capillaries, resulting in loss of resolution and shortening the life of the capillary array.
Capillary electrophoresis is especially susceptible to salt in samples, either from template preparation, from cycle sequencing reactions, or from precipitation methods using salts. The negative ions in salts can be preferentially injected into the capillary array during electrokinetic injection, potentially leading to lower signal and shorter reads.
Many DNA preparation methods for sequencing require the recovery of DNA from lysed bacterial cultures. Unless DNA is carefully purified, protein can remain in the DNA samples. Protein can be injected and adhere to the walls of the capillary array, adversely affecting data resolution and capillary array lifetime.
Residual RNA that is present in DNA template preparations competes with the DNA for injection into the capillary array. Residual RNA has the same effect as excess salt, that is, decreased signal and shortened read lengths.
Some methods of template preparation, such as the Thermomax method for M13 preparation, use detergents such as Triton X-100 to lyse the protein coat of phage particles. Other detergents, such as sodium dodecyl sulfate (SDS), are used in plasmid purification protocols to lyse bacterial cells. Small, negatively charged detergents may be preferentially injected over DNA during electrokinetic injection. Present at high levels, detergents such as Triton X-100 and SDS will adversely affect the life of the capillary array and the quality of the sequencing data.
Quality control parameters as outlined by Applied Biosystems 3730/3730xl DNA Analyzers Sequencing Chemistry Guide Part Number 4331467Rev. B