NanoBio: Plasmid Verification

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Contents

Growing up more plasmid DNA

  1. Touch a sterile plastic pipet tip to chosen colony (only one colony!).
  2. Dip then swish tip in 5 mL LB media containing 5 µL Amp (100 mg/mL in distilled water) in a 14 mL tube.
  3. Shake the tube at 37 °C for 12-16 hrs.
  4. Use Qiagen's Spin Mini-prep kit to mini-prep the vector from the culture. Follow the instructions provided, except elute with 30 µL rather than 50 µL.
    • The typical yield from a 5 mL culture is ~10 µg for the pSB1AC3, pSB1AK3, & pSB1AT3 plasmids.

Analytical Digestion and Gel of Biobricks Part

  • Before doing a digest, check that the size of your new part is significantly different by ~20% or 500 bp (whichever is smaller) from the vector backbone and any of the component inserts you used to construct it.
  • If the insert is greater than 100 bp in length, then you can digest with any combination of prefix (EcoRI, XbaI) and suffix (SpeI, PstI) enzymes. My default digestion is X|P.
  • If the insert is less than 100 bp, try to find a non-Biobrick/Biofusion enzyme that will cut inside your insert. Alternatively, digest with ApaLI and one of the Biobrick/Biofusion enzymes so that your insert is within a fragment of ~550 bp.
    • Mix:
      • 2 µL mini-prepped DNA (~200 ng DNA)
      • 1 µL 10x FastDigest Buffer
      • 0.5 µL FastDigest XbaI (only 0.5 units are required)
      • 0.5 µL FastDigest PstI (only 0.5 units are required)
      • distilled water to bring to 10 µL total volume
    • Run a 1-2% agarose gel to check for the presence and size of the insert.

Sequencing of Biobricks or Biofusion Part

  • Only sequence new parts, final devices, and useful intermediates.
  • Sequencing provides ~1000 bp of information.
  • Confirm the entire sequence of the BioBricks part. This may require sequencing using both forward & reverse primers.
    1. For each sequencing reaction, submit ~ 500-2000 ng of plasmid DNA and 3.2 pmol of one appropriate primer in a total volume of 13 uL.
    2. In a 1.5 mL eppendorf, mix:
      • plasmid DNA (I shoot for ~500 ng)
      • 6.5 uL 2x primer solution
      • water to a total volume of 13 uL.
    3. 2x primer solution = 3.2 pmol primer/6.5 uL solution, or 4.9 uL 100 mM primer plus 995 uL sterile water.
      • For the pSB1AC3, pSB1AK3, and pSB1AT3 vector, use AF009 and AF010 as forward and reverse primers.
      • For the V0100 or V0120 vectors, use AF039 & AF040 as forward and reverse primers.
      • For any of the Sikorski vectors, use AF058 & AF059 as forward and reverse primers.
    4. To submit your requests, fill out and print the order form. Drop your tubes off at Stanley Hall Rm 237.

Archiving the New Part

  • All new parts should be stored in the Molecular Foundry strain collection; these are the blue labeled boxes on the Common Stocks shelf in the -80C in Rm 5204. It is also helpful to keep parts that you have made in your personal strain collection.
  • For the 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 Sm920.
  • For the strain collection, grow a bacterial culture either to mid-exponential phase or overnight. Mix equal volumes 50% glycerol and culture in an autoclaved 2 mL screw-top tube. Store at -80.
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