The Ocular Molecular Genetics Institute, Massachusetts Eye and Ear Infirmary

DSCVR

The DNA Sequencing Center for Vision Research (DSCVR) currently offers automated sequencing services to vision scientists at the Massachusetts Eye and Ear Infirmary, the Schepens Eye Research Institute, and Harvard University.  It offers these services at a reduced rate to NEI-funded vision scientists from these institutions and to all vision scientists at the Massachusetts Eye and Ear Infirmary.  The cost for the sequencing is supplemented from grants from the National Eye Institute and by funds from the Massachusetts Eye and Ear Infirmary.

Vision Scientists interested in setting up an account at DSCVR are asked to contact us at DSCVR@meei.harvard.edu.  To facilitate our annual reports to the NEI, users will be asked to provide the NEI number(s) of the grants whose work which will be aided by this service.  All principal investigators will be provided with a username and password which will enable them to retrieve their sequence results via ftp (see below).

CONTACT INFORMATION:

Physical location: 4^th floor, 325 Cambridge Street, Boston

Address for U.S. Mail:      DSCVR

                                            c/o Terri McGee

                                            243 Charles Street

                                            Boston, MA 02114

Fax:      617-573-3168

Terri McGee 617-573-3284

Maria Janssian 617-573-3038

It is critical that DNA samples are of sufficient quantity and quality for the sequencing reaction to work properly. Poor sequence data is often due to poor quality DNA.  The following are guidelines for preparing your DNA samples for the DSCVR.

1. All samples (mixtures of template and primer) must be suspended in sterile distilled H₂O (dH₂O), and not in tris-EDTA (TE) buffer.  EDTA will inhibit the sequencing reaction.
2. All samples must be free of salts and ethanol.  Recommended purification methods are below.

It is difficult to estimate low concentrations of plasmid DNA or PCR products using a spectrophotometer.  We strongly advise that the DNA concentration of samples be estimated by running a known small volume of the samples on an agarose gel adjacent to a DNA mass ladder (e.g., Invitrogen cat. #10068-013 or #10496-016).

After amplification, an aliquot of the PCR product should be run on an agarose gel to estimate the concentration of the sample and to ensure that the product is a single band on the gel.  If nonspecific products are present, it is likely that one must adjust the PCR conditions to eliminate these extra bands to obtain quality sequence, or, alternatively, it is necessary to gel-purify the correct band.

The DNA templates must be purified prior to sequencing to remove excess PCR primers and reagents.  We recommend using either the QIAquick PCR purification kit sold by QIAGEN (cat #28104 or #28106) or the PCR clean up kit sold by Roche (cat #1696 513).

For sequencing, supply the DSCVR center with 8 ml containing 1-2 ng per 100 bases of purified PCR product and 3.2 picomoles of the appropriate sequencing primer resuspended in sterile dH₂O. Remember to include only one primer per reaction!  If the sequence in both the sense and antisense direction is desired, one must submit two template/primer samples for a total of two separate sequencing reactions.

For the best sequencing results from plasmid DNA, we recommend reamplifying the plasmid insert by PCR and purifying this PCR product as described above prior to sequencing.

If you want to ignore this advice and sequence the plasmid DNA directly, we recommend that you use plasmid DNA that is isolated from minipreps using one of the recommended kits or methods below.  For sequencing, supply the center with 8.0 ml of a solution containing 200-500 ng of purified double-stranded plasmid DNA (50-100 ng if single-stranded) and 3.2 picomoles of sequencing primer resuspended in dH₂O.

Suggested plasmid purification kits:

QIAGEN QIAwell Plus Plasmid MINI Kit

QIAGEN Plasmid MINI Kit

Promega Wizard Plus MINI Prep

Avoid primers with long stretches of a repeated base (over 3-4 bases).  Avoid primers with repetitive or palindromic sequences that may self-hybridize.   Standard primers based on sequences flanking insert sites in plasmids (such as T3 and T7) should work well for templates derived from plasmids.

Before submitting samples, users must first provide DSCVR with an electronic list of the samples being submitted.  This is done by filling in and submitting the Sample Submission Form.  In the top portion of the form, fill in your name, your email address and the name of the principal investigator of the lab.  In the fill-in box list the names of all of the samples being submitted.  Use a carriage return following the name of each sample. 

Please use the following naming convention for your samples:  initials assigned to PI_template-name_primer_tube number (or well position for 96-well plates).  For example: a sample from Ted Dryja’s lab with a template from DNA sample 001-201 and an M13 forward primer, provided in tube # 4 would be labeled TD_001-201_M13F_4.  It is important not to include commas, spaces, or slashes in sample names.  Use underscores to separate the user initials, template, primer, and tube number.  If you are providing samples in a 96-well plate, provide the well position in place of a tube number.  For example the same sample in well position B9 would be named, TD_001-201_M13F_B9.  This naming convention and format is important so that we can keep track of samples from the many investigators who will be using this facility.  Sequence files generated by the ABI3100 machines will contain these names so users will have a record of the template and primer attached to the results.

When the form is complete, click the "Submit Request" button.  The form will automatically be emailed to the DSCVR with the time and date of submission attached to the email. We will email a confirmation when we receive your form.  This will serve as your record of what was submitted.   Samples will be sequenced generally on a first-come first-served basis.  Samples will not be sequenced until the DSCVR Submission Form is received.

Each submitted sample must be in its own tube and must contain the purified template and primer already mixed at the concentrations specified above and in a volume of 8 ml.  Each tube must be labeled with the tube number referenced on the DSCVR Submission Form for that sample.  Include with your set of tubes your name, principal investigator's name and the date that the DSCVR Submission form was submitted.  This will help us to link the samples with the correct form.

If you prefer, you can submit your samples in 96-well plates.   Each well must contain 8 ml of the template/primer mix as described above. On the plate cover please write your name,  principal investigator's name, the date that the DSCVR Submission form was submitted.  On the DSCVR Submission form, indicate the name of the samples in wells A1 through H12.  If you are submitting more than one plate on a given day please submit one DSCVR Submission form for each plate.  Include both on the plate and in the form a unique identifier so that we know the plate that matches each submission form.

Deliver the samples to the DSCVR facility located in the Ocular Molecular Genetics Institute on the 4^th floor of 325 Cambridge Street.  This building is owned by the Massachusetts Eye and Ear Infirmary, but it is not the main building.  It is very close to the Charles Street T stop on the Red Line.  We try to have someone always present on the 4^th floor to accept your samples during regular weekday hours.  There is a doorbell located above the phone which is on the wall to the right of the elevators (as you exit).   If nobody is present to respond to the doorbell, leave the samples in the mail slot located on the wall next to door labeled freezer room.  Please note that this building and the 4^th floor are accessible only from 7:30 am to 5:30 pm Monday through Friday.  AT OTHER TIMES THE BUILDING IS LOCKED, AND, EVEN IF YOU MANAGED TO GET INTO THE BUILDING, THE ELEVATOR ACCESS TO THE 4^TH FLOOR IS RESTRICTED AND YOU WILL NOT BE ABLE TO GET TO THE 4TH FLOOR.

Once the sequencing reactions are completed, the sequence files will either be uploaded to the appropriate principal investigator's password-protected site on the DSCVR server or emailed directly to the user according to the users preference.  Users can access these files via ftp using programs such as FTP Explorer or FETCH. The ftp address for the server is:  65.112.7.90.  When logging onto this ftp site, you must enter a user name and password.  Each principal investigator will be assigned a user name and password; please contact us ( DSCVR@meei.harvard.edu ) if you don’t have a password or if you’ve forgotten it.  All members of a principal investigator’s lab will share the same name and password.  .  We will send each investigator an email notifying him/her when new sequences have been placed on the web site.  The output requires software for analysis and we expect that most users will have software for viewing the chromatograms.  If you do not have the software required for analysis, and if you expect to be analyzing only a few sequences, contact us and we may be able to let you use software in our lab to view your data.  If you don’t have the software and you are anticipating that you will be analyzing a large number of sequences, we suggest that you download free software or purchase commercial software for this purpose.  Software for viewing the data is available from the following sites:

The software package VectorNTI, facilitates viewing, sequence alignment and more vigorous sequence analysis and can be obtained free of charge for academic institutions from Invitrogen. https://catalog.invitrogen.com/index.cfm?fuseaction=userGroup.home.

Other software packages can be purchased (e.g., from Applied Biosystems, Sequencher, LaserGene, etc.)

Researchers will be charged $3.50 per sequence. Vision scientists at the Massachusetts Eye and Ear Infirmary will receive an additional $1.50 discount ($2.00 per sequence) because of additional subsidy available to members of the MEEI Ophthalmology department from an NEI-department core grant and department support.   It is possible that in the future these rates will change.  You will be charged whether or not the sequence of a sample actually worked, unless the problem with the sequence was due to difficulties at DSCVR itself. 

We plan to send out bills to the principal investigators at the end of each calendar month.  To pay an invoice, researchers at the Mass. Eye and Ear can indicate a cost center number to be charged.  Researchers at either the Schepens Eye Research Institute or Harvard University must send a check made out to the "Massachusetts Eye and Ear Infirmary" to our address (see top of page).