sources AddGene, Odin and https://jemi.microbiology.ubc.ca/sites/default/files/Chang%20et%20al%20JEMI-methods%20Vol%201%20pg%2022-25.pdf =Grow starter colony of DH5alpha -make 1 plate LB Agar(@35g/liter) so 10ml water + .35g LB Agar microvawe repeatedly and wait 1 hour to cool down -streak the plate with DH5 bacteria from a stab to get a starter colony -incubate 12 -24 hours at 37 Celsius until you get 1mm/+colonies. Result -> A starter colony =Make plates for post transformation recovery and amplification -make 1 plate of LB Agar AMP by using a big glass bottle with 10ml water + .35g LB Agar microvawe repeatedly and wait to cool to 60 Celsius (can hold in hand without burns). If using the ODIN kit, use the provided tube that has 6g LB Agar AMP to disolve in 100 ml or 150 so it should be 0.6 to 0.4 g /10ml LB Agar and Amp for a plate so it's enough for 10 plates. If making from scratch (having the advantage of sterilization) add 10ul of Amp 1000x by (using the calculator spreadsheet) to the big glass bottle and let cool for 1 hour -make 1 positive control plate plain LB Agar by using a big glass bottle with 10ml water + .35g LB Agar, microvawe repeatedly and wait 1 hour to cool down. -Mark the plates (LB Agar Amp and transformed DH5a SELECTIVE), and (LB Agar and GFP transformed DH5a) and store them for later steps. =Preparing recovery LB media - Take a new CT and add 0.02 grams dry LB and 1 ml distilled water to a the microcentrifuge tube and shake vigurously to dissolve the LB. -Microvawe and let cool to room temperature This will be your post shock recovery media. - label CT "final GFP transformed bacteria" OR If using the ODIN kit: -fill one of the ODIN LB powder microcentrifuge tubes with 1 ml of room temperature water -shake and dissolve the LB ==Transformation =Make competent bacteria 3.1 Using an inoculation loop, gently scrape many bacteria off of your fresh plate until the loop is filled, and mix it into the “bacterial transformation buffer (about 100ul PEG800_CaCl2) tube. Mix until any big clumps have disappeared. This might require gently sucking the mixture up and down using a transfer pipette. Your transformation mix should be very cloudy, if not repeat the first step until the liquid turns hazy but not quite opaque in the tube. You can store these competent cells at 4 Celsius in the fridge for 1-2 days if you are not immediately performing the experiment. We suggest attempting one experiment at a time which gives you multiple opportunities to repeat the experiment.) Result ->almost full centrifuge tube called bacterial transformation buffer containing DH5 and Buffer =Add Plasmid -Get tube labeled plasmid and (force DNA droplets to bottom of tube by holding closed cap end and flick (https://www.youtube.com/watch?v=5XbLrXtWNOk) with index downwards) -Using your pipette, add its contents to the competent cell mixture tube labelled bacterial transformation buffer. Gently mix by flicking the bottom of the tube with your finger a few times. =Shocking -Incubate this tube in the fridge or on ice or ice water for 30 minutes using foamy rafts if incubation is a water. -Incubate the bacterial transformation buffer” for 30s in 42 Celsius water by placig the bottom 2/3 in heated liquid. You can approximate this temperature by using water that is warm, but comfortable enough such that you can still keep you hand in it. -Return the tubes to remain in the ice water for 2 minutes. (optional according to ODIN) -Using the same pipette, add 1ml of liquid LB media from the "preparing recovery LB media" step to your competent cell mixture containing your DNA, and mix gently by flicking the closed tube. 3.6 Incubate the tube at 37C for 2 hour with shaking if possible or 4 hours at room temperature. This step allows to bacteria to recover and replicate the inserted DNA. Do not shorten the time, this step is key for the experiment to work. If you are having trouble with your experiment increasing this incubation time up to 12 hours will increase the chances of experimental success. MORNING -Only if the plates from make plates step in the fridge! Take all plates from make plates step and let them warm up to room temperature. -Transfer 200 ul (4-5 drops) of the solution from the tube labelled bacterial transformation buffer that should be almost full to the surface of the agar of (LB Agar Amp and Competent bacteria and Plasmid) using a micropipette . Using an inoculation loop or the spreader, gently spread the bacteria around the (LB Agar Amp and Competent bacteria and Plasmid), late and let dry for 10 minutes before putting the lid back on. - Positive Control Transfer 100 ul of the solution from the tube labeled bacterial transformation buffer to the surface of a plate with "LB Agar and GFP transformed DH5a". Using a spreader, gently spread the bacteria around the plate and let dry for 10 minutes before putting the lid back on) and half with innoculating loop. 3.8 Flip the plates upside down to prevent condensation from forming and dripping onto your bacteria. Incubate all plates except for the starter plate so the one with transformed bacteria on Amp and the one with transformed bacteria with LB Agar and No Amp at 37 Celsius for 16-24 hours or room temperature for 24-48 hours. Once you see little white round dots forming put on your glasses and shine the blue light on the Amp plate with bacteria. The glasses help block the blue light so you can see the flourescence of the Jellyfish GFP protein in the bacterial cells. Both plates should have some bacteria. The control plate should have much more bacteria because it should contain both the bacteria with the GFP plasmid and the bacteria that did not get a plasmid. If the positive control plate does not have any bacteria after waiting as long as 36 hours it means your shocking killed all the bacteria. The CT with Amp should have at least 2 colonies of GFP fluorescent bacteria. 4 Disposal and Clean-up 4.1 It's not dangerous yet it's good for the used loops, used pipettes, foam holder (if used), and microcentrifuge tubes should be disposed in a biohazard container with water and 5% bleach. The agar plate containing bacteria that you used for your initial culture should also be disposed in the same container.