BT-491 Molecular Biology Lab Manual (prepared by NKJ)

Lab-2- Molecular Biology Lab

Experiment-No.-2: Transformation of E.coli DH5α with plasmid DNA by Calcium
chloride treatment
Aim:
To demonstrate the bacterial transformation using plasmid DNA (either pUC18 or pBR322)

Theory:
Genetic transformation is a process by which a cell takes up naked DNA from the surrounding
medium and incorporates it to accrue an altered genotype that is heritable.
Natural Transformation first observed by Griffth in Streptococcus pneumoniae. The overall
process in this organism occurs in several steps as follows:
1) Development of competence
2) Binding of DNA
3) Uptake of DNA
4) Formation of pre-integration complex
5) Integration of DNA in the recipient cell chromosome or maintenance as a extra
chromosomal DNA.
In case E.coli, artificial transformation process, that is by the treatment with calcium chloride, is
used to transform it with plasmid. Because it lacks the natural competence factor in it.

Material:
Sterile Conical flask- 100 ml, Sterile test tube, sterile 1.5 ml microfuge tube, sterile glass pipette,
micro pipette, petriplate, spreader, LB medium, LB agar plate with ampicillin (100 μg/ml), 50
mM CaCl2, E.coli DH5α, Palsmid DNA (pUC18), or pBR322

Method:
a) Preparation of E. coli competent cells by CaCl2 treatment
1. Grow 10 ml overnight culture of the single colony of the E.coli DH5α strain in LB media
in a 100 ml conical at 37oC.
2. Dilute overnight culture 1/100 to 10 ml LB broth in flasks 5 - 10 X culture volume (i.e.
25 ml in 250 ml flask etc.) (i.e. add 0.1 - 0.2 ml O/N culture in 10 ml LB).
3. Grow to early log phase (OD600 = 0.3 - 0.4) (90 - 180 min depending on the strain at
37oC).
4. Chill the culture by keeping in ice for 30 minutes.
5. Collect cells by centrifugation (4000-5000 rpm for 10 min or 4000 x g for 5 min) at 4oC.
6. Keep the cells ice cold in all further steps.
7. Resuspend the cells in 1/2 culture volume (5 ml) of 50 mM ice-cold CaCl2. Hold on ice
for minimum of 30 min.
8. Collect cells as before and gently resuspend them in 1/10 culture volume (0.1 ml) 50 mM
CaCl2.
9. Keep the tubes in ice for 1 hr.
10. (Competent cells can be stored almost indefinitely by adding ice-cold sterile glycerol to a
final concentration of 10% (v/v). Mix and leave on ice for 30 min, then store at -70 oC.)

b) Transformation

1. Take two 1.5 ml microfuge tube.

2. In the experimental tube, mix 0.1 ml aliquots of cells with pUC18 DNA (1 - 10 ng)
3. In the second tube use as control, add only buffer or sterile DDW without DNA
4. Leave on ice for 10 - 30 min.

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BT-491 Molecular Biology Lab Manual (prepared by NKJ)

5. Heat shock at 42oC for 2 min. (90 - 120 s is OK).

6. Add 0.9 ml LB and allow expression for 30 - 60 min at room temperature shaker before
plating.
7. Plate 0.05 ml and 0.1 ml in LB-agar plate containing ampicillin (50-100 ug/ml) and keep
it at 37oC for over night.
8. Count the number of colonies in the experimental plate.
9. Calculate the transformation efficiency

Precautions:
Observation and results:
Discussion or interpretation:
Reference: Dagert and Ehrlich (1979) Gene 6:23-28.

Determine the transformation efficiency

Transformation efficiency is expressed as the number of antibiotic-resistant colonies per microgram

of pUC18. Because transformation is limited to only those cells that are competent, increasing the amount
of plasmid used does not necessarily increase the probability that a cell will be transformed. A sample of
competent cells is usually saturated with small amounts of plasmid and excess DNA may actually interfere
with the transformation process.

This is an example: Let you got 1000 colonies at your LA-Amp plate.

The formula is:

total number of cells growing on the LB/amp = # of transformants per ug of DNA

amount of DNA spread on the agar plate

a. Determine the total mass of pUC18 used. _____________________

( let you used 5 uL of pUC18 at a concentration of 0.1 ug/uL.)

Total Mass = volume x concentration.

b. Calculate the total volume of cell suspension prepared. _100 ul______________________

c. Now calculate the fraction of the total cell suspension that was spread on the plate.

( Number of uL spread/total volume) ____100 ul /1000 ul_____________________

d. Determine the mass of pUC18 in cell suspension. __________________________

(Total mass of pUC18 X fraction spread.)

e. Determine the number of colonies per ug of plasmid. Express in scientific notation.

Number of colonies observed/mass pUC18 spread ( from calculation in step (d) = transformation
efficiency.)

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BT-491 Molecular Biology Lab Manual (prepared by NKJ)

2. This is the transformation efficiency. What factors might influence transformation efficiency? Explain
the effect of each you mention.

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