Training report

Rajarshi Verma
Techniques and equipment learnt
• Plasmid isolation by alkaline lysis
• Gel electrophoresis
• DNA elution from gel
• DNA amplification using PCR
• Making of competent cells
• Chemical Transformation of competent cells
• Restriction digestion of plasmid
Plasmid Isolation
• The basis of this technique is that there is a narrow pH range at which non-
supercoiled DNA is denatured, whereas supercoiled plasmids are not.
• When the pH is adjusted to 12.0–12.5, then the hydrogen bonding in non-
supercoiled DNA molecules is broken, causing the double helix to unwind and the
two polynucleotide chains to separate.
• If acid is now added, these denatured bacterial DNA strands reaggregate into a
tangled mass.
• The insoluble network can be pelleted by centrifugation, leaving plasmid DNA in
the supernatant. An additional advantage of this procedure is that, under some
circumstances (specifically cell lysis by SDS and neutralization with sodium
acetate), most of the protein and RNA also becomes insoluble and can be
removed by the centrifugation step.
• Further purification by organic extraction or column chromatography may
therefore not be needed if the alkaline denaturation method is used.
Competent cell preparation
Competent cells are ready to use bacterial cells that possess more
easily altered cell walls by which foreign DNA can be passed through
easily. Most types of cells cannot take up DNA efficiently unless they
have been exposed to special chemical or electrical treatments to make
them competent .
These were prepared by following methods:
• CaCl2 Method
• Electroporation
Hanahan or CaCl2 Method

• Competency can be achieved by opening small pores in bacterial cells

by suspending them in a solution containing a high concentration of
calcium.
• The divalent cations generate coordination complexes with the
negatively charged DNA molecules and lipopolysaccharide.
• The heat shock step strongly depolarizes the cell membrane of CaCl2-
treated cells.
• Thus, the decrease in membrane potential lowers the negativity of
the cell’s inside potential which ultimately allows the movement of
negatively charged DNA into the cell’s interior. The subsequent cold
shock again raises the membrane potential to its original value.