Silver: 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.
    • The typical yield from a 5 mL culture is ~35µL of ~150ng/µL or ~5µg.

Analytical Digestion and Gel of Biobricks Part

  • If the insert is greater than 200 bp in length, then digest with the enzyme pair that you initially used to install the part into the BioBricks vector (see digestion).
  • If the insert is less than 200 bp, use ApaLI and a second enzyme (EcoRI, SpeI, etc.,) such that your fragment of interest is ~300-600 bp. Note: the basic protocol is the same as below, except replace XbaI and PstI with ApaLI and EcoRI (or SpeI, etc).
    • Mix:
      • 3 µL mini-prepped DNA (~100 ng DNA)
      • 1 µL 10x BSA
      • 1 µL 10x NEB Buffer 3 (assuming digestion with XbaI and PstI; otherwise select other appropriate buffer (see NEB website)
      • 0.1 µL XbaI (only 1-2 units are required)
      • 0.1 µL PstI (only 1-2 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 Part

  • Only sequence new parts, final devices, and useful intermediates.
    1. For each sequencing reaction, submit ~ 600 ng of plasmid DNA and 3.2 pmol of the appropriate primer in a total volume of 12 uL.
      • To confirm the entire sequence of the BioBricks part, both forward and reverse sequencing should be done.
      • For the V0002 vector, use O0007 and O0008 (alias IP047 & IP048) as forward and reverse primers.
      • For the V0100 or V0120 vectors, use O0001 and O0002 (alias CA012 & CA013) as forward and reverse primers.
      • For any of the Sikorski vectors, use O0005 and O0006 (alias DL068 & DL069) as forward and reverse primers.
      • If your final part is quite large (greater than ~1600bp), consider using O0020-22, which bind to the Kozak & tADH1 regions. See our internal database for more details.
    2. To submit your requests electronically, go to the DFCI molecular biology core website http://mbcf.dfci.harvard.edu/. Once you have created an account, select 'Enter your DNA requests.' Choose the Fast Track service, and once you have submitted the request label your tubes with the ID numbers. Drop your tubes off at the Fish room on the 3rd floor of the Smith Building.

Archiving the New Part

  • All new parts should be stored in the Silver Lab plasmid collection and strain collection. 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. Quick freeze the mixture by immersing in liquid nitrogen. Store at -80.
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