Automated DNA Sequencing is an essential service by the
DNA Sciences Core. We use the state-of -art capillary instrument, 3730 DNA
Analyzer from the Applied Biosystems. Specific nucleic acid sequences can be
determined as insert in a bacterial
phage vector (single-stranded), plasmid vector, lambda vector, BAC or YAC
(all double-stranded), or as a PCR product (direct sequencing). Genomic DNA
or mitochondrial DNA can be sequenced directly of after certain
Currently, we use the BigDye Terminator v3.1 chemistry
from Applied Biosystems, which provides top performance under Sanger
sequencing methods. Typically with a clean template with roughly balanced
bases, double stranded and plasmid DNA will yield 750-800 bp with an accuracy
of 98% or higher. Single stranded DNA can be sequenced up to the same length
or longer with the same accuracy. For direct sequencing of PCR products the
long templates can reach the similar specifications as the plasmid DNA while
the short ones can have various degrees of accuracy because of the varying
affinities they possess for the DNA polymerase due to their lengths.
Ordering a sequencing run includes two steps. First, users
are to fill out an Online Request Form.
Unnecessary delay could result in if the form is not completed properly.
The turnaround time is normally 24-36 hours
for samples submitted before 10 AM. Users may pledge their case with us if
they need expeditions for special occasions, such as a grant deadline.
For difficult-to-sequence templates we have established a
regiment of methods to deal with but we need some prior knowledge of them to
prescribe an effective recipe to use. The different approaches include the
use of DMSO or betaine, or modification of the cycling profiles. In addition,
modified nucleotides are sometimes used to alleviate the compression
problems. We encourage users to consult with us when they encounter or
anticipate difficult templates.
Other chemistries, such as dye primer sequencing, and
selected chemistry with alternate enzymes, such as Sequenase, are also
available upon request. Special arrangements can be made to switch
chemistries upon request. DNA samples can be sequenced with primers provided
by the users or requested from our Primer Bank.
The bank was built with the intention of including as many commonly used
primers as practical and will expand on reasonable suggestions.
You can now order oligos from the UVA-Invitrogen program
(See the logo in the left panel) that may save you a lot in primer costs. It
is convenient, quick, and less hassled -- You order your oligos online, NO
PTAO is required. You can pick up the oligos in the core and the get billed
only once a month, again online. You need to register to use this feature,
please contact us if you need help.
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