Competent cell preperation and bacterial transformation.ppt

Component cell preparation and bacterial transformation. ppt
Biotechnology ( plant genetic transformation) second year fourth semester of agricultural biotechnology or agricultural engineering.

1. Preparation of Competent Cells
and
Bacterial Transformation

4. Stages:

5. Step 1 Competent cell Preparation

9. What happens due to CaCl2treatment to the cells ?

11. Commercially available competent cells of E.coli: JM109 , DH5-alpha

12. Step 2. Bacterial Transformation

16. Transformation of bacteria
by heat shock
•After DNA is added to the cells, the suspension is “heat
shocked” —or moved quickly between widely different
temperatures.
•The combination of chemical ions and the rapid change in
temperature alters the permeability of the cell wall and
membrane, allowing DNA molecules to enter the cell.

18. •Transformation is highly
inefficient –only one in
every 10,000 cells
successfully incorporates
the plasmid DNA
•Since each colony comes
from a single transformed
cell, we can determine the
number of cells
transformed per
microgram of DNA
•Quick reference:
•50 ng (0.05µg) plasmid DNA
•0.5 mL recovery volume
•0.25 mL plated
Transformation Efficiency