advotech instructional video enhanced transformation protocol [Music] the bacteria will be grown for 18 to 22 hours on the lb agar Source plates and colonies will be taken from these plates by the instructor to make liquid E coli cell cultures cells will be collected via centrifugation and will be made competent using calcium chloride and competent cell solution once the competent cells have been prepared the plasmid is added and just like a traditional transformation protocol these cells are briefly heat shocked lastly recovered bacteria will be plated onto selective medium and incubated at 37 degrees Celsius overnight before visualization Step 1 preparation of source plates remove a single backdo bead from the E coli gfp host vial using a sterile inoculating Loop and place the bead on top of the agar Source plate near the edge dissolve the bead by adding 10 microliters of recovery broth streak the loop back and forth through the dissolved back toe bead to make a primary streak rotate the plate 90 degrees and streak through the primary streak once creating zigzags across a clean section of the plate continue this pattern of streaking across the plate label and cover the plates incubate inverted at 37 degrees Celsius for 18 to 22 hours Step 2 preparation of E coli starter culture to create the E coli starter culture you will need a water bath set to 37 degrees Celsius a permanent marker a sterile inoculating Loop a source plate that has been grown between 18 to 22 hours recovery broth a 50 ml conical tube and a vortexer [Music] after your water bath reaches 37 degrees Celsius add 30 ml of recovery broth to the 50 ml conical tube and label it E coli culture swipe the sterile inoculating Loop through a dense section of the bacterial culture about a match head-sized amount of bacteria resuspend by twisting the loop back and forth until all the bacteria has been removed from the loop if available Vortex the tube to resuspend the bacteria completely place the E coli culture into the 37 degrees Celsius water bath for 60 minutes during the 60-minute incubation label 1.5 mm microcentrifuge tubes E coli you'll need two tubes per student group after the 60-minute incubation aliquot one mil of the culture into each of the E coli tubes place these tubes on Ice until they are needed for the experiment these tubes can be stored up to 24 hours after aliquating before storing centrifuge tubes at Max Speed for 5 minutes and pour off the supernatant then store the cell pellet at 4 degrees Celsius step 3 preparation of competent cells each student group should obtain two tubes labeled E coli if not done already by the instructor centrifuge the tubes at Max Speed for a few minutes to pellet the cells [Music] carefully pour off or pipette out the supernatant without disturbing the cell pellet add 200 microliters of ice cold calcium chloride solution to each tube gently resuspend the cells by slowly pipetting up and down several times save the remaining calcium chloride on ice for a later step make sure the cells are fully suspended continue to gently pipette until no clumps are seen in the calcium chloride solution [Music] incubate on ice for 10 minutes [Music] centrifuge to the tubes for five minutes [Music] carefully pour off or pipette out the supernatant place the tubes back on ice add 100 microliters of ice cold competent cell solution to each tube and resuspend the cells by pipetting up and down several times place the tubes immediately on ice the confidence cells can be stored up to 48 hours in the freezer after they have been resuspended with the competent cell solution [Music] thank you step 4 transformation label one tube plus DNA and the other minus DNA [Music] add 150 microliters of ice cold calcium chloride solution to each of the tubes and mix by pipetting up and down to the plus DNA tube add 10 microliters of the plasma DNA incubate the tubes on ice for 10 minutes place the transformation tubes in a 42 degrees Celsius water bath and incubate for 45 seconds immediately transfer the tubes to ice and incubate for two minutes add 250 microliters of recovery broth to each tube invert the tubes to mix incubate in a 37 degree celsius water bath for 10 minutes [Music] while the tubes are incubating label each plate after the recovery period remove the tubes from the water bath using a one mil pipette transfer 250 microliters or the volume recommended in your protocol of the minus DNA recovered cells to each of the appropriate plates using another one mil pipette transfer 250 microliters or the volume recommended in your protocol up the plus DNA recovered cells to each of the appropriate plates spread the cells over the entire plate using an inoculating Loop place the plates in an incubation oven or leave at room temperature [Music] step 5 visualization after overnight incubation use a blue light or UV light to visualize your transformation results