Question

PREPARATION OF COMPETENT CELLS and TRANSFORMATION:

2a. We will be preparing competent MM294 (E. coli) cells using a chemical treatment of CaCl₂. What are MM294 cells, why do we use them? Define “competent” cells and describe why/how CaCl₂ makes cells “competent”. Why is “mid-log” phase important? Support your drawing with a cartoon of what cells “look like” before and after treatment.

2b. What does “heat-shock” do (in the transformation procedure)?

2c. What is LB? What does “LB” stand for and what is its role in cloning? What would happen if LB was NOT on the plates?

Answer 1

2 a. MM294 are a strain of E. coli which isolate in the lab of Matthew Meselson that's why it named as MM294. when we clone DNA in MM294 strain it gives good yields of DNA.

Competent cells are those cells that ready to transform or can accept DNA from surroundings.

CaCl2 promotes the binding of DNA to surface of bacteria. Ca++ binds with negatively charged DNA. after heat shock this DNA can enter into the cell. We use bacteria mid log phase because in this phase bacteria are rapidly dividing and after transformation chance of getting more copies of our cloned DNA is increased.

2b. When we give a heat shock (42°C) to the cell, between the outside and the inside of the competent cell a pressure difference occur. due to this pressure difference pores are formed in the cell wall of bacteria, through this pore DNA can enter inside the cell. when we put the cell in normal temperature the cell wall heal itself.

2c. LB medium is a nutritionally rich edium used for culture of bacteria. LBs stand for Lysogeny Broth. In laboratories generally we called it Luria-Bertani medium. Its compositions are tryptone, yeast extract and NaCl. In cloning experiment LB medium is used for culturing of transformed cells. If LB is not in the plate, growth of transformed cells is affected.