INTRODUCTION

A plasmid is referred to a DNA molecule that is separate, and can replicate independently from
the chromosomal DNA. They are usually circular, and are double stranded. They usually occur
naturally in bacteria, but in some instances are found in eukaryotic organisms. Plasmid sizes vary
from 1 to 1000 kilobase pairs. The number of identical plasmids inside the cell can range
anywhere from one to one thousand under some circumstances. They can be considered to be
part of the mobilome, since they are often associated with conjugation, a mechanism that takes
place during horizontal gene transfer.

Plasmids are almost always purified from liquid bacteria cultures usually E. coli which have
been transformed. Almost all plasmid vectors in common use encode one or more antibiotic
resistant genes as a selective marker, for example kanamycin and ampicillin, which allows the
bacteria that have been transformed to multiply uninhibited.

When bacteria are lysed under alkaline conditions, both DNA and protein are precipitated. The
concentration of NaOH is used at 0.1M in order to reduce the formation of ssDNA. After the
addition of acetate-containing neutralization buffer the large and less supercoiled chromosomal
DNA plasmids can renature and stay in solution.

MATERIALS AND METHODS

Genomic DNA isolation

In order to isolate genomic DNA from the cells, 1.5ml of overnight culture was pipetted into a clean
eppendorf tube, and centrifuged at 5000 rpm for 5 minutes. The supernatant was then decanted. After
that the pellet was resuspended in a 500µl of lysis buffer which contains a 100µl/mol of proteinase K .
A 500µl of protein precipitation solution ………….. was added and was vortexed vigorously for 20
seconds. The sample was then spun in a microcentrifuged for 2 minutes at 12 000rpm in order to pellet
the protein debris. The top aqueous layer was then transferred to a clean tube. A 1/10 volume of 3M
sodium acetate, pH 5.2 was added to the aqueous phase and mixed briefly by finger tapping. 2 volumes
of ice cold isopropanol was then added and mixed gently by inverting the tube several times. This was
then placed at -20 for 15 minutes. After that the tube was spun at 12 000rpm’s for 10 minutes. The
supernatant was poured and the liquid drained on an absorbent towel. 600µl of 70% ethanol was also
added, and the tube was then gently inverted several times in order to wash the DNA. The tube was
then spun for 1 minute. After that the ethanol was then poured off slowly and carefully, watching the
pellet so that it does not pour off. The liquid was poured off on an absorbent towel. The tube was then
allowed to air dry for 15 minutes. The pellet was then resuspended in 60µl of 1XTE. The concentration of
DNA was then determined.

PURIFICATION AND LIGATION OF UNKNOWN DNA INTO CLONING VECTOR

In order to carry out this part of the experiment,

BACTERIAL TRANSFORMATION

PART 1 : Transformation procedure

A 100µl of competent cells was added to each of 3 sterile 1.5ml eppendorf tubes on ice. After that
10µl of the respective ligation mixtures to each test tube. The tubes were gently swirled, and put on
ice for 30 minutes. The cells were then heat shocked by placing in a 42 0C waterbath for 2 minutes. A
1ml pre-warmed LB broth was added to each tube, and incubated at 37 0C for an hour. A 100µl, 200µl,
300µl of the cells were plated on the 3 respective LB agar plates containing 50µg/ml ampicillin, IPTG,
and X-gal, and were incubated at 370C for 24 hours, and were plated upside down. The number of
colonies and transformation efficiency was determined.

RESULSTS

TABLE 1:THE RESULTS OBTAINED UPON PLATING

PLATES OBSERVATIONS

IPTG+ X-Gal+ LB+ AMPICILLIN No growth

IPTG + X-Gal + LB No growth
IPTG + X-Gal + ampicillin No growth
LB only No growth
LB + Ampicillin No growth

DISCUSSION