Competent Cell Flashcards
What are competent cells?
They are bacteria capable of taking up DNA.
What is transformation?
It is where DNA is moved into competent bacterial cells.
Why is E. coli used often to make competent cells?
Because it is not a competent cell.
What is used to punch holes in the cells?
CaCl2
Why do the cells have to be kept on ice during the procedure?
to limit the damage that the CaCl2 causes.
How old do the cells need to be in order to undergo transformation?
they have to be young like early log phase to handle the damage.
What is moved into E.coli during the transformation procedure with a ligation mix?
the vector pACYC184 or a pACYC184 that is ligated to insert that will carry a heme transport genes of plesiomonas shigelloides
What resistance genes does pACYC184 have?
a chloramphenicol resistance gene (Cm) and a tetracycline resistance gene (Tet).
What will the ligation mix contain?
What will pACYC184/pACYC184 ligated into the insert contain?
it will have both pACYC184 and pACYC184 that has been ligated to the insert, either would carry a intact cmR gene that allows the bacteria that took up the DNA to grow on the media containing chloramphenicol
What is the first step to transformation with a ligation mix?
make the cells competent using MgCl2 or CaCl2 treatment
What is the second step to transformation with a ligation mix?
to transform the competent cells with the ligation mix
What is the third step to transformation with a ligation mix?
to plate out the bacteria on media containing chloramphenicol
Why are steps done on ice when making competent cells?
so the cells are not damaged severely by the procedure.
Why are early log cultures centrifuged when making competent cells?
so that the cells can be concentrated and also removed from the media and its waste products
