This document provides instructions for preparing competent bacterial cells for transformation. Key steps include growing cells to mid-log phase, treating with calcium chloride to make membranes permeable, adding exogenous DNA, and applying a heat shock to induce DNA uptake. The resulting transformed competent cells can be used to take up exogenous DNA from their surroundings through their membranes. Proper preparation is important for achieving high transformation efficiency.

1. PREPARATION OF
COMPETENT CELLS FOR
TRANSFORMATION
BY KANCHAN YADAV
MSC AGRIL. BIOTECHNOLOGY ,1ST YEAR
DR. RAJENDRA PRASAD CENTRAL AGRICULTURE UNIVERSITY

2. INTRODUCTION OF DNA INTO A HOST CELL
– TWO KEY PROBLEMS
• Must be able to physically cross the cell membrane
• Once inside the new host cell , our DNA must be able to
replicate

3. TRANSFORMATION
• Transformation is the genetic alteration of a cell resulting from
the direct uptake and incorporation of exogenous genetic
material from its surroundings through the cell membrane(s).
• For transformation to take place, the recipient bacteria must
be in a state of competence, which might occur in nature as a
time-limited response to environmental conditions such as
starvation and cell density, and may also be induced in a
laboratory.

4. COMPETENT CELLS
• Bacterial cells that are able to take up DNA from the
environment are called competent cells.
• In the laboratory, bacterial cells can be made competent and
DNA subsequently introduced by a procedures.

5. METHODS OF TRANSFORMATION
• USE OF CELLS WHICH ARE NATURALLY COMPETENT
EG . BACTERIA Bacillus subtilis
• CALCIUM TREATMENT OF CELLS
• TRANSFORMATION OF PROTOPLASTS
• ELECTROPORATION OF CELLS/ PROTOPLASTS
Using high voltage shocks for a fraction of a second
to make membrane more permeable to DNA

6. METHODS OF TRANSFORMATION
• MICROPROJECTILE BOMBARDMENT
Bombardment of intact cells with metal particles
coated with DNA
• INFECTION OF CELLS WITH VIRUSES
• MICROINJECTION of DNA directly into a cell nucleus

7. HEAT SHOCK TRANSFORMATION
• Heat shock transformation uses a calcium rich environment
provided by calcium chloride to counteract the electrostatic
repulsion between the plasmid DNA and bacterial cellular
membrane.
• A sudden increase in temperature creates pores in the plasma
membrane of the bacteria and allows for plasmid DNA to enter
the bacterial cell.

8. MATERIAL REQUIRED
• Buffers and solutions
• 1) CaCl2.2H2O ( 1M)
• 2) Standard transformation buffer(TFB)
• 3) MgCl2 – CaCl2 solution
• 4) Ice cold
• Media
• 1) LS or SOB medium for initial growth of culture.
• 2) SOB agar plates containing 20 mM MgSO4 and the
appropriate antibiotic
• 3) SOC medium

9. MATERIAL REQUIRED CONTD.
• Nucleic Acids and Oligonucleotides
• Plasmid DNA
• Centrifuges and Rotors
• Sorvall GSA rotor or equivalent
• Equipment
• Polypropylene tube (50mL), chilled in ice
• Polypropylene tubes
• Water bath preset to 420C

10. PROCEDURE
• 1. Inoculate the E. coli culture into the LB medium and incubate at 37
°C for 24 h with vigorous shaking at 180 rpm.
• 2. Aliquot 0.5 ml of the grown culture into 50 ml of LB in a 200-ml
conical flask. Prewarm the broth to 37 °C.
• 3. Incubate at 37 °C with shaking at 180 rpm.
• 4. Monitor the growth regularly till the OD600 reaches to 0.35–0.4.
• 5. When suitable growth has been reached, chill the culture on ice.

11. PROCEDURE CONTD.
• 6. Transfer the culture to an autoclaved centrifuge tube and collect the cell
pellets by centrifugation at 6000 rpm for 5 min at 4 °C. Discard the
supernatant.
• 7. Resuspend the cell pellets in 20 ml of an ice-cold 50-mM CaCl2 solution.
Incubate the resuspended cells on ice for 20 min.
• 8. Collect the cell pellets by centrifugation at 6000 rpm for 5 min at 4 °C.
• 9. Resuspend the cells with 2.5 ml of ice-cold 50-mM CaCl2. Optionally, if
required to store the competent cells for a longer period, resuspend the
cells with 2.5 ml ice-cold 50-mM CaCl2 containing 10 % glycerol.
• 10. Use 100 μl of the prepared competent cells for transformation.
• 11. Dispense the competent cells into aliquots of 100 μl and store them at −
80 °C for further use.

12. CONCLUSION
• Non-commercial preparations should normally give 106 to
107transformants per microgram of plasmid;
• a poor preparation will be about 104/μg or less,
• but a good preparation of competent cells can give up to
~108 colonies per microgram of plasmid.
• Protocols, however, exist for making supercompetent cells that
may yield a transformation efficiency of over 109.

13. CONCLUSION CONTD.
• The chemical method, however, usually does not work well for
linear DNA, such as fragments of chromosomal DNA, probably
because the cell's native exonuclease enzymes rapidly degrade
linear DNA.
• In contrast, cells that are naturally competent are usually
transformed more efficiently with linear DNA than with plasmid
DNA.
• The transformation efficiency using the CaCl2 method
decreases with plasmid size

14. PRECAUTIONS
• 1. CaCl2 is a hazardous material for skin, eyes and the respiratory
system and may cause burns. Hence, use gloves while using the
same.
• 2. Avoid thawing of cells before use.
• 3. Keep cells on ice not longer than 3 h; do not use cells again that
have been on ice.
• 4. You may stock-freeze the competent cells in liquid nitrogen. The
stock-freezing might
• 5. Keep cells viable for a longer period, but it decreases the
transformation efficiency by at least a factor of ten.