Lab – Transformation – Virtual
Transformation
The ability of bacteria to take up
Lab – Transformation – Virtual
Transformation
The ability of bacteria to take up DNA fragments from the environment and ultimately express the information encoded in the genes of that DNA is termed transformation. Transformation has profound implications for antimicrobial chemotherapy because the genes that are taken up by transformation may be genes that code for antibiotic resistance. In this exercise we will transform a strain of E. coli that is normally susceptible to ampicillin into a resistant strain.We use chemical transformation not electroporation.
We use chemical transformation not electroporation.
The video below is a better video which goes over the theory of transformation as well as a transformation experiment in the lab.
https://dnalc.cshl.edu/view/15916-DNA-transformati…
https://dnalc.cshl.edu/resources/dnatoday/2020-vir…
Plasmid for the Virtual Experiment
This plasmid pEGFP is a genetically modified version of the Green Fluorescent protein gene which produces a blue fluorescent protein (see below). It also contains an ampicillin resistance gene for selectability.
Virtual Experimental Procedure
Collect two sterile epi tubes and label one “+” and one “-“.
With a sterile 1mL pipette transfer 0.25 mL of ice cold 0.05M calcium chloride into each tube.
Place both tubes on ice in a beaker.
Take tubes to front bench. Instructor will pipet 20 uL of competent cells into each tube. Mix the contents of each tube. Return the tubes to the ice.
On ice bring the tube labeled “+” to the front of the class and have 2 uL of plasmid DNA (pEGFP) added to the “+” tube.
NO DNA will be added to the tube labeled “-“. Mix the contents of the tube well and return it to the ice. Allow both tubes to sit in ice for 10 minutes.
Collect 2 LBA/Amp plates and label one “LB/AMP + plasmid” and one “LB/AMP – plasmid”.
Collect 2 LB plates and label one “LB + plasmid” and one “LB – plasmid”.
Heat Shock both tubes by submerging the bottom ½ of the tube into the 40-42 C waterbath for 90 seconds. (Heat shocking allows the DNA to more readily enter the cells)
Place both tubes back on ice for at least 1 minute.
Add 0.5 mL of Luria broth (LB) to each tube, according to your instructor’s directions, and let both tubes stand at 37 C for 15 minutes.
Transfer 0.2 mL of “+ plasmid” tube to the plate labeled “LB/AMP + plasmid”.
Transfer 0.2 mL of “- plasmid” tube to the plate labeled “LB/AMP – plasmid”.
Open the slim end of the foil package and retrieve one sterile plate spreader.
Transfer 0.2 mL of “+ plasmid” tube to the plate labeled “LB + plasmid”.
Transfer 0.2 mL of “– plasmid” tube to the plate labeled “LB – plasmid”.
Spread the culture over the “LB/AMP + plasmid” plate spreader across the surface. Repeat with the same spreader on the “LB + plasmid” plate.
Incubate all plates at 37 degrees C for 48 hours.
