Method: Toothpick Plasmid Assay

April 25 1990

Matthew S. Holt

Purpose:

The toothpick assay is used to identify bacterial colonies containing recombinant plasmids following transformation and to obtain an approximation of the plasmid insert sizes.

1. approximately 16 hours to grow isolated bacterial colonies
2. 1/2 hour or more to pick the colonies (depending upon the number)
3. exactly 20 minutes incubation
4. minimum of 3 hours to run a gel; the results are clearer at 5 hours

Special Reagents:

1. Toothpick lysis buffer
2. 0.8% TBE agarose gel

1. Prepare a 0.8% TBE horizontal agarose gel with enough wells for each colony and control to be tested. Have ready an LBM+ antibiotic plate marked on the bottom with a labeled grid to serve as the master plate Prepare eppendorf tubes labeled for each colony to test as well as for controls. Use a pipet tip (or autoclaved toothpick) to scrape the plasmid-containing E.coli cells from one single isolated colony. Smear the cells on the bottom of an eppendorf tube and immediately touch the tip to the corresponding spot on the master plate. Place the master plate at 37 degrees C. Prepare the following control tubes: 1) if available use a colony from the transformation control plate and 2) use 100 ng of the vector plasmid.
2. Add 30 ul Toothpick Lysis Buffer vortex and incubate for exactly 20 minutes in a 65 degrees C waterbath. Longer incubations sometimes results in doubling of all bands. After incubation vortex the samples and spin for 15 seconds in a microcentrifuge. Load the samples and controls including a lane with 1 kb ladder (BRL). A large amount of cells results in a fairly viscous solution. Be careful not to load any bubbles which will pull the sample out of the wells. It is not necessary to load all 30 ul to visualize the bands.
3. Run the gel in 1X TBE buffer at 80 volts for a minimum of 3 hours. Better separation of different recombinants are observed with a 5 hour run. The gel can run at higher voltage provided the buffer is well circulated (Dias pumps are not adequate for this). If the buffer warms up the results are distorted.
4. Stain for at least 30 minutes in approximately 1L ddH2O with 40 ul ethidium bromide destain in ddH2O for 20 minutes and photograph. Colonies that contain a recombinant plasmid will have bands higher than that of the vector-only control. Prepare a small scale plasmid prep of the insert-containing clones by inoculating the appropriate amount of LBM+ antibiotic directly from the master plate. If the bacteria are barely visible on the master plate take a small plug with the small end of a sterile pasteur pipet from the correct area and use as inoculum.

1. Toothpick Lysis Buffer

   1. 1.25 ml 1N NaOH
   2. 0.25 ml 0.5M EDTA pH 8.0
   3. 0.625 ml 10% SDS
   4. 1.75 g Ficoll
   5. 250 ul 1% Bromophenol blue dye

   Adjust volume to 25 ml with dH2O. Filter sterilize with a syringe filter and store at -20 degrees C in 1 ml aliquots. Can be repeatedly thawed without harm.

2. TBE (Tris-borate/EDTA buffer)

   5 X TBE Stock:

   1. 54 g Trisma base
   2. 27.5 g boric acid
   3. 20 ml 0.5 M EDTA (pH 8.0)

   Adjust volume to 1L with dH2O. Store in a glass bottle at room temperature to avoid precipitation and discard any bottles that develop a precipitate.

References:

Sambrook J. Fritsch E.F. and T. Maniatis.(1989). Molecular Cloning A Laboratory Manual. Second edition. Cold Spring Harbor Laboratory Press Cold Spring Harbor NY. p. 1.32.