NanoBio: Archiving Your Plasmid in the Strain Collection
Archiving the New Part
- All new strains should be listed in our electronic and physical strain collections. It is also helpful to keep parts that you have made in your personal strain collection.
- It is recommended that you also add new plasmids to the plasmid collection. In case the -80C freezer ever completely defrosts, the plasmid backup will allow you to easily remake your strain.
- the strain to be archived
- a mini-prep of the plasmid contained in this strain
- appropriate media, usually LB
- 50% glycerol in water, sterile
- Cryotubes, 1.8 mL volume, catalog #
Adding a Strain to the Electronic Strain Collection
- Open Plasmid_Cell Stock Registry in Excel.
- Give your plasmid a name.
- Plasmids are names Lxxxx, where L is a letter and x is a number.
- The letter indicates the function of the part. Currently
- V=Vector Backbones
- C=Binding Proteins
- D=Delta (Knockout strains)
- E=Reporter Proteins
- G=Genetic Recombination Parts
- H=Thermo Stability
- I=Electron Transfer Proteins
- K=RBS's, translational parts
- L=terminators, transcriptional modifiers
- P=Promoters, binding sites
- S=Structural proteins
- Z=Combination Parts
- Use the next available number within a letter-sequence. For example, the next binding protein part after C5201 should be C5202.
- Save the sequence of the plasmid in the Clone Sequences folder on Nanofiler. The filename should be the name of the plasmid, e.g. C5202.gb for part C5202.
- Check that the next available MFe# has not already been used. To do so, use Ctrl-F (find) to see if that MFe# is already somewhere else in the spreadsheet.
- Give the strain containing your plasmid a MFe#. Use the next available # as indicated at the top of the spreadsheet.
- Update the next available #.
- Insert an entire row (not just a series of cells or else you throw off the entire spreadsheet) in the spreadsheet within the appropriate section, i.e. C for Binding proteins.
- Fill in all the requested information into the spreadsheet
- id#: the MFe number, e.g. MFe030
- author: your initials, e.g. C.A-F
- plasmids: the names of all plasmids in the Lxxxx format, e.g. T5015
- brief 'part' description: VAAL-E4 peptide
- vector backbone: the plasmid backbone, using the same name found in the vector section, i.e. pSB1AC3
- Cell Line: the name of the cell strain, e.g. Top 10
- More complete description:
- size(bp): the size of just the part
- biofusion or biobricks part?: if your part is in biofusion or biobricks format, please indicate here
- VectorNTI file: the name of the vector NTI file, which should be the same as the plasmid name, i.e. T5015.
- other notes:
- Refs.: If this is described in a published paper, please insert that citation here.
- Save the spreadsheet and close it so that others can use it.
Adding a Strain to the Physical Strain Collection
- For the physical strain collection, grow a bacterial culture either to mid-exponential phase or overnight. Mix 900uL 50% glycerol and 900uL culture in an autoclaved 2 mL screw-top tube. We call this a glycerol stock of bacteria.
- Label the tube lid with the MFe#. Label the side of the tube with the MFe#, the plasmid name, and the antibiotic resistance carried by the plasmid.
- Place the bacteria-containing glycerol stock in the appropriate box (labeled strain collection) in the Common Stocks shelf in the -80C freezer.
- Recommended: Make another bacteria-containing glycerol stock for your personal stock collection and store it in the -80C freezer.
- Place 5 uL mini-prepped plasmid DNA in a labeled eppendorf tube in the appropriate plasmid collection freezer box in the -20 freezer in Rm 5204.Label the side of the tube with the MFe#, the plasmid name, and the antibiotic resistance carried by the plasmid