NanoBio: Archiving Your Plasmid in the Strain Collection

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Contents

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.

Materials Needed

  • 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

  1. Open Plasmid_Cell Stock Registry in Excel.
  2. 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
      • J=Transporters
      • K=RBS's, translational parts
      • L=terminators, transcriptional modifiers
      • P=Promoters, binding sites
      • S=Structural proteins
      • T=tags
      • 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.
  3. 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.
  4. Give the strain containing your plasmid a MFe#. Use the next available # as indicated at the top of the spreadsheet.
  5. Update the next available #.
  6. 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.
  7. 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.
  8. Save the spreadsheet and close it so that others can use it.

Adding a Strain to the Physical Strain Collection

  1. 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.
  2. 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.
  3. Place the bacteria-containing glycerol stock in the appropriate box (labeled strain collection) in the Common Stocks shelf in the -80C freezer.
  4. Recommended: Make another bacteria-containing glycerol stock for your personal stock collection and store it in the -80C freezer.

Plasmid Collection

  • 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
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