We currently run all samples submitted to the Low Throughput Lab (LTL) on our ABI 3700 using dye terminator chemistry. You can expect to receive between 400 and 800 high quality bases
We will run all samples submitted
one time. We will rerun some sequence
reactions that, for unknown or unapparent reasons, produce short reads or an
unusual number of ambiguous base calls that we think we can improve by running
the sequence reaction again. We will
also repeat sequence reactions that appear to completely fail on the first
run. These reactions produce few
(<100 High Quality Bases) and it is unknown if the reaction failure was due
to an error setting up the reaction or a primer-template problem. Please do not expect us to rerun samples submitted
to us when: the template concentration was too low, the volume of the sample
submitted prevented us from measuring the template concentration and then
setting up the sequence reactions properly or your samples contained enough
ethanol to inhibit the sequence reaction.
Template Concentration:
Template concentration should equal or exceed 100 ng/ul. We can get good sequence from less concentrated samples, but there is more variability. There are no guarantees we can sequence samples that measure less than 50 ng/ul. Samples that are submitted at less than 50 ng/ul will be amplified and then run one time. Expect shorter read lengths (350 – 600 HQB) and less overall quality in these amplified plasmids.
We use 2 ul of each sample
submitted to measure the template concentration on our fluorometer. In general, the DNA concentrations measured
on our fluorometer have been approximately 40% less than the
concentrations measured by our users who determine [template] on a
spectrophotometer. So adjust your
estimates accordingly. Unless you have
submitted enough samples to make specific adjustments of your own, subtract
40% from your OD readings.
How much template is needed? Plasmids
Nanograms of template needed for each
sequence reaction
Divide the total number of bases
(plasmid + insert) by 10 to give the nanogram amount of template needed for
each sequence reaction. Multiply by the
number of primers you are using and then double it. If we have 2x the amount of template necessary, reruns can be set
up for the next sequencer run and turn-around time for your sequence is
significantly reduced. We also use 2 ul of each sample to check the concentration on
the fluorometer, so plan accordingly.
We will keep your unused templates and primers for 3 months. After 3 months they will be discarded.
How much template is needed? PCR Products
Divide the total number of bases in your fragment by 10 to give the nanogram amount of template needed for each sequence reaction. Multiply by the number of primers you are using and then double it. If we have 2x the amount of template necessary, reruns can be set up for the next run and turn-around time for your sequence is significantly reduced. We also use 2 ul of each sample to check the concentration on the fluorometer, so plan accordingly.
How much template is needed? Cosmids and BACs
The more template you provide
the better the results will be. We will
need 1.0 to 3.0 ugms of template per sequence reaction. We will bill you double the cost of
plasmid/PCR sequencing for cosmids and BACs.
See us before you get started.
Please submit all custom primers between 5.0 and 10.0 uM. We will use 1.0 to 2.0 ul / reaction. Melting temperature of these primers should be between 45 and 600C.
Would you like to get your DNA sequence without a
mini-prep?
Get DNA sequence from bacterial cells in liquid culture or from colonies picked from agar plates.
Bring 5 to 10 micro liters of a saturated bacterial culture grown in LB media supplemented with the appropriate antibiotic. Please, submit cultures grown in LB media only. “Extremely rich growth media” may interfere with the amplification phase of this procedure.
OR
A small amount of material from a colony may be picked from an agar plate, resuspended in 20 micro liters of sterile TE (10 mM Tris, 1 mM EDTA, pH 8.5) and then submit to the LTL for sequencing. Please be careful and not transfer any agar with the colony.
OR
If the clones are required for
further use:
Pick a colony from an agar plate and resuspend it in 20 micro liters of sterile LB supplemented with the appropriate antibiotic. Submit 5 micro liters to the LTL. Please be careful and not transfer any agar with the colony.