Protein Biotechnology with GFP MegaLab - Module 1

Added: 2026-05-05

[00:00:02]advotech instructional videos presents exploring biotechnology with the green fluorescent protein module 1 transformation of E coli with gfp in module 1 you will transform the gfp host E coli bacteria with the p fluorogreen plasmid the host bacteria will be grown for 18 to 22 hours on lb agar Source plates collected using a sterile Loop and made competent in calcium chloride next the gfp plasmid will be added to half of the cells before they are briefly heat shocked finally the bacteria will be allowed to briefly recover before they are plated onto lb auger plates and incubated at 37 degrees Celsius overnight for this module you will need a container of ice holding tubes of calcium chloride and P fluorogreen plasmid three sterile inoculation loops a tube containing recovery bra plus an empty microcentrifuge tube and finally lb agar plates that are either plain plus ampicillin or plus ampicillin and iptg step one label the microcentrifuge tube containing ice cold calcium chloride as minus DNA and the empty microcentrifuge tube AS Plus DNA step two using a sterile inoculation Loop transfer approximately five well-isolated colonies from the E coli Source plate to the minus DNA tube step 3 twist the loop between your fingers to free the cells resuspend the bacterial cells in the calcium chloride Solution by pipetting up and down until no clumps of cells are visible and the cell suspension looks cloudy treat the cells gently step 4 transfer 250 microliters or half of the cell suspension to the tube labeled plus DNA Place both tubes on ice [Music] step 5 add 10 microliters of P fluorogreen plasma DNA to the tube labeled plus DNA and gently flick the tube to mix do not add plasmid to the minus DNA tube step 6 incubate both tubes on ice for 10 minutes step 7 float both transformation tubes in a 42 degrees Celsius water bath for exactly 45 seconds step 8 immediately return the tubes to the ice bucket and incubate for two minutes [Music] step 9 carefully transfer 250 microliters of recovery broth to each tube using a sterile one mil pipette gently mix by flicking the tubes step 10 incubate the cells for 10 minutes in a 37 degree celsius water bath Step 11 while the tubes are recovering label the bottom of four auger plates as follows label the plate with no stripe as minus DNA label the plate with a single stripe as minus DNA plus amp and finally label both striped blades AS Plus DNA plus amp Plus iptg step 12 after the recovery period remove the tubes from the water valve Step 13 using a sterile one mil pipette transfer 250 microliters of the recovered cells from the tube labeled minus DNA to the middle of the minus DNA and the minus DNA plus amp plates step 14 using a new sterile one mil pipette transfer 250 microliters of the recovered cells from the tube labeled plus DNA to the middle of the plus DNA plus amp and the plus DNA plus amp plus iptg plates step 15 spread the cells over the entire plate using an inoculating Loop [Music] use one sterile Loop to spread both minus DNA samples change to a fresh Loop before spreading the plus DNA samples make sure the cells have been spread over the entire surface of the plate cover the plates and wait five minutes for the cell suspension to be absorbed by the auger step 16 stack the plates on top of one another and tape them together label the plates with your initials or group number once you are sure that the liquid has been absorbed into the agar place the plates in the inverted position auger side on top into a 37 degree celsius bacterial incubation oven for overnight incubation step 17 after the overnight incubation visualize the transformation and control plates using a long wave UV light you are now ready to proceed to module 2.