Toronto/Lab Protocols


Overview of Lab Protocols

1. Transformation via Heat Shock (protocol here)

  • Insert genes of interest (in plasmid form) into E. coli. Plate and grow.

2. Mini-prep Overnight (MP O/N) (protocol here)

  • Increase the quantity of desired DNA by growing clones of the bacteria that have them in a test tube.

3. Mini-prep (MP) (protocol here)

  • Extract genetic material from bacteria.
  • Extracted material will be in plasmid form.

4. Plasmid Digestion (PD) (protocol here)

  • Cut the genes of interest free from the plasmid with the use of restriction enzymes.
  • The first PD will be a sample run. A small quantity will be done and run on a gel (gel refers to agarose electrophoresis) to make certain that the desired genes are present (and that your enzymes are working).
  • The second PD will be a full PD, using a larger quantity (use the previous PD to gauge plasmid concentration, and use this to estimate the quantity of MP needed). Run on a gel using the 1kb ladder.
  • Using the transilluminator, cut out the relevant genes from the gel. MAKE CERTAIN TO CUT ON THE CUTTING BOARD.
  • You cannot stop at the PD stage. You must go straight into Gel Extraction.

5. Gel Extraction (protocol here)

  • Extract the DNA from the gel (that you cut it out in step #4) using the extraction kit. Your end product will be purified DNA.

6. Quantitation (protocol here)

  • Determine the concentration of the purified DNA (which is in solution).
  • Run another gel (a small sample run) along with three wells of HindIII ladders of varying concentrations. You will have one additional well of 1 kb ladder.
  • You will contrast the brightness (not lengths) of the ladders vs. your DNA.

7. Ligation (L) (protocol here)

  • Using the data gathered from #6, you will use this step to:
    • Ligate any number of genes together
    • Reinsert genes into a plasmid

8. Transformation via Heat Shock

  • You get to do the cloning process all over again, with your “new” gene sequence. Have fun.

note: These protocols, along with all supply protocols (such as making LB, agar plates, and competent cells) can be found in hard copy in the lab.