Transformation

Question 1

What is transformation

Answer 1

-introduction of recombinant DNA molecules into host organisms
-cells that are able to take up added
DNA are called COMPETENT CELLS

Question 2

methods of transforming DNA into a cell

Answer 2

CaCl2
-partially disrupts the cell membrane (permeable),
allowing DNA molecules to enter (most common)

In vitro packaging
-infecting cell extracts containing all the
components to assemble intact phages. Most common vectors used
are λ Phage and cosmids. Infection of host cells with these vectors
result in a very efficient injection of recombinant DNA.

Electroporation
-treat cells with a short burst of electric current to make them more permeable

Ballistic Transformation
-DNA attached to microscopic particles can be physically
“shot” into cells.

Question 3

equation for transformation efficiency

Answer 3

(number of transformants/ug of DNA) * [final volume at recovery (mL)/volume plated(ml)] = Number of transformants per ug

Question 4

What do blue colonies indicate?

Answer 4

cells transformed with cloning
vectors not containing inserts
(β-galactosidase is active)

Question 5

What do white colonies indicate?

Answer 5

cells transformed with recombinants
(β-Galactosidase gene disrupted
by insert )