LAB 1: UV MUTAGENESIS AND PERCENT SURVIVAL

OBJECTIVE/AIM:
To evaluate the effect of UV irradiation on bacterial cell proliferation.

PRINCIPLE:
Ultraviolet (UV) light kills cells by damaging their DNA. The light initiates a reaction
between two molecules of thymine, one of the bases that make up DNA. The resulting
thymine dimer is very stable, but repair of this kind of DNA damage--usually by excising or
removing the two bases and filling in the gaps with new nucleotides--is fairly efficient. Even
so, it breaks down when the damage is extensive. The longer the exposure to UV light, the
more thymine dimers are formed in the DNA and the greater the risk of an incorrect repair or
a "missed" dimer. If cellular processes are disrupted because of an incorrect repair or
remaining damage, the cell cannot carry out its normal functions. At this point, there are two
possibilities, depending on the extent and location of the damage. If the damage is not too
extensive, cancerous or precancerous cells are created from healthy cells. If it is widespread,
the cell will die.

EXPERIMENTAL PROCEDURE:
1. Dilute culture of E. coli in 500 ml of LB broth and aerate at 37oC overnight.
2. Prepare fresh LB broth, inoculate 1000 ml of LB with 1000 ul E. coli culture and incubate
at 37oC incubator shaker. Incubate for 4-5 hrs. until log phase reached.
3. Measure the OD at 600nm. Required OD is 0.4 to 0.5. Do not take cell if OD is more than
0.6.
4. Divide the 500 ml culture in autoclaved conical flask according to the time points.
5. Keep one flask in white light and other flask under UV light for required time point, for
example, for 5 mins, 10 mins, 15 mins, 20 mins up to 60 mins.
6. At every time point take the O.D of white light culture and UV irradiated culture at 600
nm. Note down the readings and plot graph for percent survival.

RESULTS AND CONCLUSION:

Time point (minutes) White Light O.D UV O.D
0
5
10
15
20
 25

Plot the bar graph based on the reading and discuss how different UV exposure has different
effect on bacterial cell proliferation.

PRECAUTIONES:
1. Always wear gloves.
2. All glass wares and pipettes should be autoclaved.
3. Should not be exposed to UV.
4. Always work in laminar hood.