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FACILITY INFORMATION
Facility Director
Anne V. Kane, M.D.
akane@tufts-nemc.org
Technical Staff
Lynn Gorczyca
Location
Tupper Building, Room 822 15 Kneeland Street
Tufts-New England Medical
Center
Mailing Address
750 Washington Street, Tufts-NEMC
Boston, MA 02111
Telephone
617-636-7611 |
We provide services and reagents, at competitive prices,
for molecular biology and microbiology research. Our services
are available to all Tufts scientists.
Preparation of Plasmids
Our philosophy is that plasmid preps can be performed
more efficiently, consistently, and cost effectively by
a central facility. Because these are done in the core
on a daily basis, we have experience hard to march in individual
labs. All the critical steps in the procedure—the
little tricks, the danger points—have been worked
out, and this results in the highest possible yields from
even low copy number plasmids.
Please note:
In order to be equitable in providing access to services, we will
receive no more than four plasmids at a time from any lab. Any submissions
greater than four will be held in reserve and completed at the discretion of
the lab director. Thanks.
Method
Our standard method is an RNAse-free alkaline lysis/double cesium gradient protocol.
Cesium gradients are less expensive than ion exchange column methods and often
give markedly higher yields, particularly with high copy number plasmids that
exceed the binding capacity of the gel. The second gradient results in a cleaner
product with much higher transfection efficiency, but it can be waived if this
level of purity is not required. The core also has established a protocol for
recovery of intact plasmid DNA from large (>40 KB) constructs such
as adenovirus. Plasmids are delivered resuspended in sterile TE buffer, with
documentation of OD 260/280 and DNA concentration.
Ordering
Plasmids are ordered by delivering a plate to
the core lab. Plates must be clearly labeled with the name of the construct,
the antibiotic required, the PI and cost center to be charged for the
procedure, and the name of the person to call when the plasmid is ready.
Once a plasmid is received, a frozen glycerol stock is prepared, so
subsequent requests for that plasmid can be made by phone or e-mail.
Turnaround time for orders is about two weeks.
In addition, the core has assembled a collection of frozen glycerol stocks
of strains containing frequently used plasmids, some of which are standard vectors;
some are for generation of fusion proteins, and some are for reporter
genes. These plasmids may be ordered from the core by phone at the
same cost as a standard plasmid prep.
Services
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