LAB Report on Plasmid Isolation, containing Principle, Protocol and Discussion

1. [LAB REPORT]
Microbial Genetic Engineering : MIC 401
SUBMITTED TO: DR. ZEENAT JAHAN
SUBMITTED BY: MOHAMMAD AL MAHMUD-UN-NABI
DEPARTMENT OF MATHEMATICS AND NATURAL SCIENCES

2. Date: 24th November, 2014
Name of the experiment:
Isolation of plasmid DNA
Purpose:
To isolate plasmid from a bacterium that was transformed with the pGLO plasmid.
Principle:
A plasmid is a small DNA molecule within a cell that is physically separated from a
chromosomal DNA and can replicate independently. They are most commonly found in
bacteria as small (usually 1 kb to 500 kb in size) and are circular, double-stranded DNA
molecules; however, plasmids are sometimes present in archaea and eukaryotic organisms. In
nature, plasmids carry genes that may benefit survival of the organism by developing
antibiotic resistance gene (R Plasmid) and production of restriction enzymes, carries genes
for utilization of some unusual metabolites, toxin production, nitrogen fixation, conjugation
and some have no apparent function (cryptic plasmids). The plasmids use the enzymes and
proteins for replication encoded from the host chromosomal DNA. If a cell contains 10 to 100
copies of plasmid are called high copy number plasmid and it the plasmid number is in
between 1 to 4 is said to be low copy number plasmid. The plasmids can frequently be
transmitted from one bacterium to another or even of another species via horizontal gene
transfer. Artificial plasmids are widely used as vectors in molecular cloning. The basic
feature of plasmid which is essential for a high quality vector-
1. The small size of plasmid is necessary to transfer larger sized exogenous DNA.
2. The unique restriction sites in plasmid help to insert the foreign DNA into the
plasmid.
3. The plasmid also contains some selectable markers or the markers may be inserted in
order to confirm the transformation of the exogenous gene.
Plasmids serve as important tools in genetics and biotechnology labs, where they are
commonly used to multiply or express particular genes. The first task of the genetic
engineering of plasmid, it needs to be isolated form the cell. In our plasmid isolating
experiment we will follow the modified alkali lyses method of Birnboim and Doly. We have
been supplied with a inoculate of a single colony of E. coli (Dh5-Alpha) to a 50ml LB broth
containing amplicilin antibiotic. The strain is resistant to ampicillin. The culture is grown for
overnight.

3. Meterials and Methods
Media:
50ml LB broth medium containing Ampicillin as antibiotic.
Reagents:
Solution 1: Alkaline lyses solution (pH 8) [Containing- 50mM Glucose + 25mM Tris +
10mM EDTA]
Solution 2: Detergent for lyses [1% SDS + 0.2N NaOH]
Solution 3: To maintain pH Balance [3M Potasium acetate + 5M Glacial Acetic Acid]
TE Buffer: [10mM Tris HCl + 1mM EDTA]
Ice cold ethanol (95%)
Equipments:
Micropipette, inoculating loop, sterile tips, Eppendorf tubes, falcon tubes and markers.
Procedure:
1. One of the bacterial colony was selected and inoculated in 50ml of LB broth
containing ampicillin antibiotic and incubated for overnight at 37°C
2. The culture was kept in room temperature.
3. For harvesting the culture, they were transferred 4 different falcon tubes (12.5ml
each) and were centrifuged at 8000rpm for 5min. the supernatant were discarded and
the pellets were collected.
4. The pellets were re-suspended to 100μl of solution 1 from all the falcon tubes and
were transferred to an Eppendorf tube. It was kept for 5min in room temperature.
5. After that 200μl of solution 2 was added to the Eppendorf tube and kept in ice cool
solution for 10min.
6. Followed by 150μl precooled solution 3 was added to the Eppendorf tube and kept for
5min at ice-cooled temperature.
7. Then the Eppendorf tube was mixed by vortex machine to make a uniform mixture
then they were centrifuged for 5min with 14000rpm at temperature 25°C. The
supernatant was collected in another Eppendorf tube and the pellet was discarded.
8. Two volume of ice-cooled 95% ethanol was added to the supernatant and was kept in
room temperature for 10min.
9. The Eppendorf tube was again centrifuged at 14000rpm for 5min and now the pellet is
collected.

4. 10. The pellet was washed and dried with 70% ethanol solution.
11. The pellet was re-suspended with 50μl TE buffer and can be stored at -20°C for future
use.
12. The Isolated plasmid DNA has to be now tested by GEL Electrophoresis with a
known ladder for comparison.
Observation:
Figure 1- Gel Electrophoresis of Plasmid DNA
L- Ladder
A- Plasmid sample (10μl)
B- Plasmid sample (5μl)
L A B

5. Discussion:
We were able to isolate the plasmid DNA form the supplied E. coli (Dh5-Alpha) sample.
After the gel run we have seen a single band for both sample A and B. But there might be any
problem occurred in the ladder DNA we have used for comparing the plasmid. The ladder
should have two distinct bands after the gel run, but we can observe only one band and it has
travelled a lot of distance, which is a bit unusual. Therefore, we cannot further analyse our
plasmid with the help of the ladder. Sample A contains 10μl of isolated plasmid and sample
B contains 5μl, therefore, the concentration of plasmid was more in sample A which resulted
a very sharp band in the gel than sample B.