Bacterial transformation consists of a bacteria cell uptaking a preprogrammed plasmid
. The plasmid consists of a vector
that always contains a segment of DNA
that expresses antibiotic resistance
. This is both beneficial to the bacteria that uptakes the plasmid and also provides a means of identifying and isolating the cells that have successfully uptaken the desired plasmid. The vector plasmid is usually cleaved at one of the MCS (Multiple Cloning Site
) by a unique restriction endonuclease
and then a piece of DNA is inserted. Vector and DNA ligate
together to form a new plasmid now expressing the newly inserted DNA (hopefully in the right direction) as well as the antibiotic resistance and any additional gene expression encoded in the vector.
A general protocol for bacterial transformation involves heat shocking and ice shocking the bacteria alternately. Note: You can also use Ca(2+) or electrophoretic waves, but I find this to be easiest to use. An example of heat/ice shocking transformation protocol is as follows:
1. Fetch a tube of frozen bacterial cells (various strains of e. coli are most popular) from the -80C refrigerator.
2. At your bench, warm the tube with your fingers until the cells just begin to thaw and place on ice.
3. Add 10 ul of plasmid DNA to approximately 100 ul of bacteria (if the DNA is not supercoiled).
4. Mix by briefly by finger flicking tube then incubate on ice for 30 min.
5. Heat shock the cells by placing the tube in a 37C incubator or water bath for 45 seconds and return to ice.
6. After 2-3 min, add 0.5 ml LB (Luria-Bertani) broth without antibiotics, mix, and incubate at 37 C for 1 hr with shaking.
7. After incubation, shake tube and aliquot 100 ul (or as much as 500 ul) of the suspension into the middle of an LB/ampicillin (use the appropriate antibiotic depending on the vector used) plate as a puddle.
8. Spread by evenly spreading cells over the plate using a “hockey puck” spreader or giant tooth pick. Be sure that you have dipped your spreader in ethanol and flamed it before spreading or that the tooth pick has been autoclaved.
9. Incubate at 37C overnight with the agar side up and the lid side down (don't forget to mark the plate properly).
The next day you should see nice little seperate colonies and then you will be ready to pick clones and see which colonies have the correct insert! What excitement!!