Plasmids
Only plasmids of traceble origin and identity should be used for any experiments. Any plasmid should be sequenced-verified with full coverage of the relevant transgene.
Procurement and cloning
We do not provide specific guidance for cloning, but make sure that all cloning is properly described in your labbooks. The initial elements used for cloning (e.g., a plasmid or a part of the insert) and the final plasmid shall be tracable and identifiable. Therefore, you should make sure that we have all material transfer agreements (or emails stating that no MTA is needed) or documentation of origin (e.g. gene synthesis by a certain company, addgene, etc.) in our database. The final product of your cloning should be properly stored and sequence verified. The palsmid maps and sequencing results (the consensus alignment) should be stored in our database.
Validation
Include in our database also notes on how the end product was validated (e.g. construct expresses well, it localizes where it should, indicating in which cell line the initial validation was performed). Plasmids
Storage
Any plasmid should be stored in your boxes and idenfied with cryogenic labels. Utilize only printed cryogenic labels (Cat. # EC1F-072WH; clean template). Currently, we use DYMO LabelWriter450 and DYMO-compatible cryogenic labels from LabTag (they are pricey, so be careful not to use them for other purposes). On the top of the 1.5ml tube we usually use, add a dot-label with printed just the code of the plasmid. You can add any other writing with a marker, but the dot-label is mandatory. On the side of the vessel, information should be printed both as QRcode and text. These precautions are needed to identify plasmids in case of damage to the labels after long-term storage.
short_name is any identifier you wish to use (can be omitted); P- indentifies plasmids; ZW is the user two letters id, or the lab id and ### is a progressive number. ZW### thus points to a record in the respective databases. XY is the user two letters id of who has prepared this stock and DDMMYY is the date. XYDDMMYY points to a record in lab books with any additional information might be stored. CONC is the cocentration with clearly identifiable units. The information in the large label (short_name excluded) is also stored in the QR code.
Intermediate cloning steps are exempt from this policies, and tubes should be disposed as soon as unnecessary.
Any plasmid should be reported in our central database, with maps and sequencing results provided in the central repository, annotations of the origin of the plasmids or the materials used to clone it (MTAs/CDAs should be specified, including AddGene) any other notes of possible use for other users.
No experiment should be run with unvalidated plasmids. No unvalidated plasmid should be ever stored in the central database. You can trust plasmids that are within the central database, but – even in this case – you are invited to sequence verify even these plasmids every time you are going to engage in a substantial undertakings (e.g. engineereing stable cell lines) or after odd-behaviours of cell lines (e.g. mislocalization of a construct).