Molecular
Genetics/DNA Sequencing and
SNPlex
Services
The
Molecular Genetics Core Facility
(MGCF),
provides automated DNA
sequencing
centered around an ABI 3130XL
Capillary sequencer.
Additional equipment housed by
the facility include a
gel based ABI
377 DNA Sequencer, an MJ Research
thermocycler, a PE Applied Biosystems GeneAmp PCR System
9700 thermocycler,
and a NanoDrop ND-1000
Full-spectrum UV/Vis
Spectrophotometer.
The MGCF also
provides Genotyping and SNPlex
services. Our methodology
is based around the
Applied Biosystems SNPlex
Genotyping System, and
is used in conjunction with ABI
3130XL instrumentation.
This combination allows for the
rapid analysis of up to 48
seperate SNPs per individual
sample. Please contact for
further details and pricing.
9,000-11,000 independent
sequencing reactions are performed per year at a cost
of $9.50 per template sequenced.
Turn-around time
Under
normal conditions sequencing
data from a template submitted
before noon on day one, will arrive
by email by the end of day two; however, the service is
first-come, first-serve, so there may be occasions where
resulting samples may take
slightly longer. The ABI
3130 capillary sequencer allows
us greater flexibility in
running samples, thus
minimizing turn-around times.
Individual investigators
provide the purified plasmid or PCR template
in water, as well as
the primer, unless a standard primer (T3, T7, M13, etc.)
stocked by the Core Facility is requested
(View
the list of available primers).
Avoid
placing samples in TE buffer,
as EDTA chelates metal ions
needed during the PCR reaction.
The sequencing reactions are performed by cycle sequencing
using fluorescent dye-labeled dideoxy terminators (ddNTPs)
and a modified Taq polymerase. Resolution of the
sequencing products is achieved by running the spin
column-purified samples on a 16 set
cap. Special
care must be taken by customers
to provide as clean a DNA
product as possible.
Quality, as well as
quantitiy of your DNA sample
is of the utmost importance. Electrokinetic injection of
the DNA product puts
investigator DNA in direct
competition with any salts, RNA,
or other charged particles which
may be present in the sample.
Detection of the fluorescently-labeled
fragments is done as the bands migrate
past the CCD camera under
illumination of an argon-ion
laser.
Version
5.2 Sequencing Analysis software,
using
optimized basecalling and
sizecalling algorithms,
is used to generate sequencing
data. Turnaround times are quick, and researchers are
rapidly provided with their sequence data via email.
Color-print electropherograms showing the fluorescence
peak traces that represent the
actual dye-labeled fragments as
they leave the
caps can be provided; however,
it is more cost- and time-efficient to simply send you the
chromatogram as an attachment to the emailed sequence
file, and we encourage investigators to use this option,
which provides a permanent electronic record which can
either be viewed on-screen or printed
at a later date. We recommend ABI's
Sequence Scanner Software v1.0
as this is
the trace viewer utilized by the
core facility for printing
chromatograms. Since the
ABI program only runs in the
Windows environment, alternative
trace viewers are available for
other platforms (Finch TV
is
available for Windows, MacOSX or
UNIX and
4 Peaks is available for
MacOSX).
Portion of a typical
electropherogram (Click to view full image).
Repeat
Policy
Sample
repeats are provided upon
customer request. We
require the sample come from the
original tube provided to the
Molecular Genetics Core
facility. If repeated results are in
conflict with the original, the
original, as well as the
repeated sample are done at no
cost to the customer. If
the repeated sample mimics the
original, both reactions are
chargeable. This is why it
is important to supply the core
with 800ng total DNA when
submitting plasmid samples, and
double the amount needed for PCR
products, see "DNA Sequencing
Order Sheet Instructions".
Quality
Assurance
An ABI pGEM
3Zf(+) control provided in every
ABI Big Dye Terminator v3.1
Ready Reaction Sequencing kit is
run regularly, as well as a Big
Dye Terminator v3.1 Sequencing
Standard kit plate to ensure the
instrument performance.
Eligible
Users
As with the other Section of
Research Resources Core Facilities, the Molecular Genetics
Core Facility serves numerous investigators at the College
of Medicine, as well as investigators outside the
University.
DNA Sequencing
Order Sheet Instructions
Template ID:
use the laboratory P.I.’s
initials and then number each
DNA sample sequentially with
every submission, e.g. JS10,
JS11, JS12 etc. The template ID
should also be used to label the
tube of DNA submitted, on the
top of each tube. If
the same DNA is being tested
with more than one primer, you
may put the DNA in a single
tube, but it must be labeled
with more than one Template ID
number, e.g. JS10-13
**Template
concentration:
SAMPLES MUST BE IN WATER.
For PLASMID and BAC
samples, the DNA
concentration should be
~100ng/µl - please submit
800ng (=8 µl at 100ng/µl) of
DNA per sequencing reaction.
For PCR products, the DNA
concentration should be
~10 ng/µl, and you need to submit
5ng PCR product
DNA/100bases length.
(Please indicate SIZE of the PCR
product in the Template size
column above; also indicate in
the Notes column that the
template is PCR product.)
Primer Name:
Indicate here the name of the
primer to be used for
sequencing. Concentrations
should be 5-10uM.
(The Core Facility provides at
no charge common primers such as
T7, T3, SP6, etc. - see full
list of available primers at
http://www.hmc.psu.edu/core/dna/primers.htm.)
If you are
using your own primer(s), the Tm
should be between 55 and 60°C;
thus, the length should be
approximately 18-25 nucleotides.
The core uses the following
equation to estimate Tm: Tm =
2oC X (A + T) + 4oC
X (G + C).
Notes:
should be used for three
purposes: 1) you may write your
own notes for reference here, 2)
request any special reaction
conditions required eg. DMSO to
minimize 2°
structure in GC rich templates,
3) indicate if the DNA template
is anything other than plasmid
DNA, eg. BAC, PCR product, etc.(
for BAC or PAC samples provide
either primer Tm or actual
sequence for Tm determination).
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