1. isolated mRNA for protein A from rat liver 2. reverse transcribed mRNA to DNA 3. added “sticky ends” to create an insert 4. ligated the insert (rat protein gene) and the plasmid (bacterial) to make a recombinant plasmid BIOTECH LAB – UNIVERSITY OF OTTAWA In this lab you will be removing a recombinant plasmid from bacteria, digesting it with restriction enzymes (endonucleases) and visualizing the result of the digestion using electrophoresis. Since the digestion takes a while you will begin the lab by setting up the digest, then while it’s running you will isolate the recombinant plasmid for the next group to use. The recombinant plasmid was prepared for you by 3rd year biochemistry students. The steps below take you through the preparation and the lab, in a logical sequence: LEADING UP TO THE LAB

WHAT YOU ARE DOING A. Isolation of Recombinant Plasmid DNA (Miniprep) 1. Pellet the bacteria, remove their growing “soup” 2. Resuspend the pellet and add lysis buffer to break open the bacterial cells. 3. Neutralize the lysis buffer using K-acetate. 4. Spin to pellet down cell debris. The plasmid will stay in the supernatant. Transfer the supernatant to a fresh microcentrifuge tube. 5. Add alcohol (just like banana DNA extraction!) to force the DNA to clump together. 6. Spin to pellet down the plasmid. Rinse with alcohol. 7. Add nuclease-free water to resuspend. Your resuspended pellet should contain the recombinant plasmid. Now, we’ll need to check for its presence! bacterial pellet supernatant (growing media/”soup”) cell debris pellet supernatant (contains plasmid) lysed (broken) bacterial cells recombinant plasmid alcohol/ broken DNA recombinant plasmid in solution

WHAT YOU ARE DOING B. Restriction Endonuclease Digestion 1. You will add restriction enzymes (aka restriction endonucleases) to the recombinant plasmid in solution. 2. Once you’ve added the enzymes the recombinant plasmid will fall apart into the original bacterial plasmid and the insert C. Gel Electrophoresis 1. To visualize the insert and the plasmid you will use gel electrophoresis. After making the gel your sample, containing the linear bacterial plasmid and the gene for rat Protein A (the insert) will be loaded on the gel. 2. The gel will act as a molecular sieve, separating the DNA by size. You will be able to see the DNA under UV light thanks to a UV-visible dye that incorporates itself into the DNA. You will receive a photograph of your DNA and a ladder that allows you to tell its size. Look carefully to locate the plasmid and the insert! recombinant plasmid in solution