When to submit to the plasmid repository
1. All unique clones produced in the labs must be fully verified by DNA sequencing and
submitted for proper storage within two months of sequencing.
Basically, the rule of thumb is that if you care enough to sequence a clone, that clone is important
enough that it should be retrievable in a timely fashion with sufficient documentation to reproduce it.
2. Clones obtained from outside sources must be fully sequenced verified and entered into
the repository within two months of receipt.
It is understood that for clones obtained elsewhere, it may not always be possible to supply all
pertinent information. Clones from commercial sources or consortia that have guaranteed the
sequence need not be re-sequenced.
How to enter a construct into the repository
Manager: Matthew Barrett; matthew_barrett@med.unc.edu
The manager is required to reject any submission to the repository that does not conform to the
following instructions.
1. Produce a new record.
a. Open relevant databases.
Data must be entered from the host computer only. Normally, FileMaker and relevant databases
are open. If not, launch FileMaker and using the current password for user = “sondek” open (File
> Open recent) three databases: Sondek plasmids, DNA sequences, and Protein sequences.
b. Create a new plasmid record.
While in the window for Sondek plasmids, start a new record (Records > New Record or Ctrl+N).
The Lab ID field will be filled and unchangeable. Fields for Location Box and Location Slot will
be inaccessible.
c. Enter relevant information for all available fields.
It is the exceptionally rare field that is left blank. For your convenience, many of these fields have
associated pull-down lists that will appear upon initial selection and a filled sample form is shown
below. Filled fields change color from red to clear.
Information on specific fields:
Gene or insert - Enter only the gene name. Please use symbol font as appropriate.
Mutation - Mention all substitutions, deletions, truncations; often used to indicate domain
boundaries.
Original source - If the template derives from an outside source, e.g., another lab or
company, pleased enter here.
GenBank ID - Often ignored but must not be left blank.
Cloning strategy and description of mutations - Provide methodology used to create the
clone including sites of gene insertion, size of insert, and how specific primers were used for
cloning. Also provide information describing protein expressed, including its borders, potential
tags and protease cleavage sites.
Primers used for cloning and mutation - Please list specific primers by their “Primer
Number” that accompany the product sheets (e.g. for Invitrogen, these numbers are found in the
upper right of each oligonucleotide record).
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Comments - typical comments include potential conflicts with GenBank entries or unavoidable
destruction of flanking restriction sites during cloning.
d. Create an associated DNA sequence record.
While in the window for DNA sequences, start a new record.
Fill the field immediately adjacent to Lab ID with the exact identifier automatically populated within
the associated Sondek plasmids record started earlier. The remaining field at the top of the record
should automatically populate with the gene name from the associated Sondek plasmids record.
If this action does not occur automatically, please do so manually.
The majority of the record is a large container reserved for sequence information. This
information should initially be formatted (Courier, 12pt, All capitals) and saved in Microsoft Word.
From Word, copy the text to the clipboard (Ctrl+C) and paste into the container (Ctrl+V).
If possible, this sequence information should encompass regions flanking the open reading
frame. The user must resolve all ambiguities in the electrophoretigram before submission. If not
possible, more sequencing is warranted to resolve conflicts. The coding region and flanking DNA
should never contain sequence ambiguities (i.e., “N”). At a minimum, bold the start and stop
codons.
e. Create an associated protein sequence record.
While in the window for Plasmid sequences, start a new record and repeat as before for entry of
DNA sequence information. Bold the start and stop of the expressed protein, as well as, any tags,
protease recognition sites, or similar information within the sequence file.
f. Verify that the three record files are linked.
View the newly created record within the Sondek plasmids database. To the right are two action
buttons: DNA sequence and Protein sequence. Click on these action buttons and verify that the
corresponding records within the DNA sequences and Protein sequences databases are
activated.
2. Store datasheets on primers
a. A copy of all cited product sheets for oligonucleotides must be added to the common three-ring
binder labeled OLIGONUCLEOTIDES and organized by date of manufacture.
3. Store DNA with record
a. Place at least 30 µl of DNA (>100 ng/µl) into a cryo-vial specifically used for the long-term storage
of small volumes.
b. Label the cryo-vial. Three distinct labels need to be applied. First, label the lid with a “Toughspot” annotated with the Lab ID. Second, marked the tube directly with the Lab ID. Third,
annotate a “Tough-tag” with the name of the clone and the parent expression vector and place on
the cryo-vial. An example of a labeled cryo-vial is near the labels and next to the computer that
functions as a server for the DNA database.
c. Give a hardcopy of the record and the corresponding DNA to the repository manager.
You cannot change already existing files in the database.
If you make a mistake in a record and save the record you will need administrative privileges to
correct the record. Please do not delete records – it messes up the numbering. If a mistake is made,
please see the manager of the repository.
Please note that this DNA bank and database will serve as our only common repository.
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Therefore, to maintain the integrity of these reagents, only the repository manager is allowed access
to the repository and individual requests must be processed through the manager.
How to obtain a construct from the repository
Only the manager of the repository is allowed access to the repository. To request a clone, print the
record and give it to the repository manager who will return the form with the requested DNA.
Amplify the clone for your personal use. Repeated requests for the same clone should not be made.
If the requested stock is low, the manager will ask that you replenish it using the guidelines above.
DNA sequencing guidelines
For primer-mediated site directed mutagenesis
Initially sequence no more than four clones and restrict the sequencing to a single pass spanning the
immediate site of the direct mutation. Once, the desired mutation is confirmed, fully sequence the
entire open reading frame in preparation for deposition of the clone into the repository. In general,
the frequency of primer-directed mutagenesis is greater than 50% and no more than four clones will
need to be sequenced to identified a useable clone. Repeat this step once more if necessary.
However, if a usable clone is not found within the first eight candidates do not continue and reinvestigate the mutagenesis strategy and redo the PCR.
For PCR-mediated subcloning
Initially sequence no more than four clones and restrict the sequencing to a single pass. Once a
potentially correct subclone is identified, fully sequence the entire open reading frame in preparation
for deposition of the clone into the repository.
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