15 Mlo Lab Report

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Label one sterile 15 mL tube “+plasmid” and the other tube “-plasmid.” You will also need to label the four agar plates as follows: LB/Amp +plasmid, LB/Amp
-plasmid, LB +plasmid, and LB -plasmid. Using a sterile pipette transfer 250 µL of ice-cold calcium chloride to each tube and place both tubes in a beaker filled with ice.
Transfer E. coli from the starter plate to the “+plasmid” tube by using a sterile inoculating loop. Spin the loop in the tubes’ solutions to make sure that all of E. coli’s cell mass is completely in the solution. The solution should turn into a milky white color. Afterwards, return the tube to the iced beaker. Repeat the same transfer process for the “-plasmid” tube. Add a loop of plasmid DNA to the “+plasmid” tube only using a clean and sterile inoculating loop and use the same loop to stir the plasmid with the cells. Do not add any plasmid DNA to the “-plasmid” tube. Put the “+plasmid” tube back in the ice and incubate both tubes for about 15 minutes. After 15 minutes, heat shock the …show more content…

Using your finger, gently tap the tubes to mix the Luria broth with the cell suspension. The test tubes will need to rest for approximately five to fifteen minutes in a room temperature environment.
Transfer 100 µL of cells from the “-plasmid” tube to the agar plates marked with “LB/Amp -plasmid” and “LB -plasmid.” Take an inoculating loop and bend one of the ends into a 90 degree angle so that the loop now resembles a hockey stick. Use the bent inoculating loop to spread the cells along the plate’s surface while making sure that you are not piercing the agar’s surface. Repeat the same process when transferring cells from the “+plasmid” tube to the plates labeled “LB/Amp +plasmid” and “LB +plasmid.”
Once all of the steps above have been completed, stack the plates upside down and wrap them securely with tape. Place the stack of plates in the incubator and incubate for 24-36

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