TECHNIQUES IN DNA TECHNOLOGY

INTRODUCTION

A plasmid is an extra-chromosomal DNA molecule separated from the

chromosomal DNA which is capable of replicating independently of the
chromosomal DNA. In many cases, it is circular and double-stranded. Plasmids
usually occur naturally in bacteria, but are sometimes found in eukaryotic.
Plasmid size varies from 1 to over 200 kilobase pairs (kbp). The number of
identical plasmids within a single cell can be zero, one, or even thousands under
some circumstances.

Electrophoresis is a procedure which enables the sorting of molecules based on

size and charge such as plasmid separation. Using and electric field, molecules
can be made to move through a gel made of agar. The molecules being sorted
move through the space in gel material. The gel is placed in an electrophoresis
chamber, which is then connected to a power source. When the electric current is
applied, the larger molecules move more slowly through the gel while the smaller
molecules move faster. The different sizes of molecules form distinctive bands on
the gel.

EXPERIMENT 7.1
Plasmid Extraction of Gram-Negative Bacteria

OBJECTIVES

i. To prepare a cell lysate

ii. To elude the plasmid DNA from Gram-negative bacteria.

MATERIALS

Sample
100 mL Escherichia coli culture

Apparatus
1.5 mL microcentrifuge tubes
Centrifuge
Micropipette and tips () 1000L,200 L,20L
Incubator shaker
-20℃ freezer
Universal bottle
Ice box
pH meter
Glassware and labware
Various sizes of latex gloves
Chemicals
Glucose
Tris-Cl
Ethylene diamine tetraacetic acid (EDTA)
Sodium hydroxide (NaOH)
Sodium dodecyl sulfate (SDS)
Potassium acetate
Glacial acetic acid
sdH2O
Ethanol 96% v/v, 70% v/v (cold)
Nutrient broth media
Ice cubes
Tris/Borate/EDTA (TBE)
RNase
Solution A (Glucose 50 mM; Tris-Cl 25 mM; EDTA 10mM)
Solution B (NaOH 0.2 N; SDS 1% w/v)
Solution C (Potassium acetate 5 M, glacial acetic acid)

METHODS

Preparation of E. Coli Culture

1. Bacteria culture was inoculated in 100 mL of nutrient broth a day before the
experiment.
2. The culture was incubated for about 8-24 hours, at 200 rpm and 35℃

Preparation of Working solutions

1. The working concentrations of solution was calculated using M 1V1 = M2V2 and
Table 7.1 was filled in.
2. The working concentration of solutions was prepared by referring Table 7.1.

Plasmid Extraction

1. The bacteria culture has been shaken in the shake flask and 1 mL has been
transfered to a clean and steriled microcentrifuge tube. The microcentrifuge
tube was labeled as A.
2. The microcentrifuge tube was spun at 10000 rpm in the centrifuge for 1
minute.
3. The supernatant was drained and the pellet was leaved in the
microcentrifuge tube.
4. The pellet was washed with 1 mL sdH2O and was mixed by tapping the
microcentrifuge tube.
5. The microcentrifuge was spun again at 10000 rpm for 1 minute, the
supernatant was drained and the pellet was leaved.
6. The pellet was resuspended 200 L withof solution A and was left for 5
minutes at room temperature.
7. Next,of solution B were added 400L and gently inversed and was put in
ice for 5 minutes.
8. After 5 minutes,of solution C 300 L were added and gently inversed and
was put in ice for 5 minutes.
9. The mixture in the microcentrifuge tube was spun at 12000 rpm for 11
minutes.
10. The supernatant in microcentrifuge A was carefully transfered into a new
steriled microcentrifuge tube without disturbing the pellet and the
microcentrifuge tube was labeled as A1.
11. The microcentrifuge A was discarded into a disposal plastic bag. The A1
microcentrifuge tube was used for further analysis.
12. Double volume of 96% ethanol was added to the A1 microcentrifuge tube.
13. Next, A1 microcentrifuge tube was kept in -20℃ freezer for 10 minutes.
14. Then, the A1 microcentrifuge tube was spun at 12000 rpm for 10 minutes.
15. The supernatant was drew off 500 L from the tube.of 70% cold ethanol
was added into the tube.
16. The A1 microcentrifuge tube was spun at 12000 rpm for 5 minutes.
17. The supernatant was discarded from the A1 tube.
18. The tube cap were opened and placed in the laminar air-flow for drying.
19. After the tube is completely 1LL dried,of sdH2O andof RNase were
20
added and mixed in the tube.
20. The tube was kept safely in -20℃ for one week to preserve the plasmid for
electrophoresis.

EXPERIMENT 7.2
Agarose Gel Electrophoresis

OBJECTIVES

i. To separate DNA fragments

ii. To observe DNA fragments
iii. To estimate the size of DNA fragments.

MATERIALS

Sample
Plasmid extract ( from previous experiment)

Apparatus
Latex gloves
Micropipette and tips () 1000L,200 L,20L
Gel electrophoresis set and power supply
UV transilluminator
Hot plate
Chemicals
Gel safe stain
x1 Tris/Borate/EDTA (TBE)
RNAase
Ice cubes
Loading dye
DNA marker 1 kb
Agarose powder

METHODS

1. An agarose gel was prepared by using dissolved 0.35 g Agarose powder and
was boiled in 50 mL x1 TBE until it is crystal clear.
2. of gel safe stain was added into 5L the dissolved gel and has been
swirled.
3. The rack and the comb was set. The was poured into the rack to solidify the
gel accordingly.
4. After 30 minutes, the comb was gently removed, the gel was put into the
case and was filled with x1 TBE until the maximum level.
5. The microcentrifuge A1 was took 1L out from -20℃ and thawed for few
minutes. Prior loading to the gel,of RNAase was added into the tube A1 and
mixed.
6. from A1 tube was transfered and 10 2 LL was added withof loading dye.
7. To load the DNA marker,from the 10 2 LL marker tube was transfered and was
added withof loading dye.
8. The sample and the DNA marker were loaded into the well gently.
9. The lid of the electrophoresis chamber was closed and current was applied.
10. The DNA bands was visualized with the UV transilluminator, the photo of the
gel was took.
11. The image were print and pasted on the column in Table 7.2 provided.
12. The DNA band samples were compared and estimated using the DNA
marker bands.

QUESTIONS

1. Plasmid band obtained was compared with the marker band. Explain the
result.

The DNA marker are visible bands of known length that are run in the
agarose gel same as the unknown samples to provide a marker for where
DNA fragment length will migrate. The DNA fragment in the plasmid band
obtained migrate at the same length as the DNA marker band.
2. Explain the precautionary steps you should engage during the extraction and
electrophoresis process.

During extraction process, make sure that the extracted DNA is treated with
RNase to remove the RNA and make sure that the vials and tips are DNase
free. During electrophoresis, wear hand gloves when handling the agarose
gel because it is slightly carcinogenic and do not touch any cooling apparatus connected
to a gel, the current can be conducted through the tubing.

3. Explain the significant study of plasmid extraction from Gram-negative

bacterium.

Extra chromosomal DNA molecule separated from the chromosomal DNA is

plasmid which is capable of duplicating independently. Antibiotic resistance,
heavy metal resistance, virulence, environmental adaptability & persistence,
and metabolic functions that allow utilization of different nutrients are some of
the known functions coded by plasmids.
RESULTS

Table 7.1: Chemical recipe for working solutions.

Chemical Molecular Stock Working Stock volume

weight concentration concentration (ml)
Solution A

Glucose 180.16 1M 50mM 0.5

Tris-Cl 121.14 1M 25mM 0.25

EDTA 372.2 1M 10mM 0.1

Top up with sdH2O 9.15

Total volume of working solution 10

Solution B

NaOH 40.0 2N 0.2N 1

SDS 10 w/v% 1 w/v% 1

Top up with sdH2O 8

Total volume of working solution 10

Solution C

Potassium acetate 5M 60 (pH 4.8) 6

Glacial acetic acid 11.5 ml 1.15

Top up with sdH2O  28.5 2.85

Total volume of working solution 100 ml 10

Table 7.2: Estimation of DNA size from E. Coli.

Image DNA marker Estimate DNA
size (kbp) size (kbp)
4 kbp Could not be
determined
DISCUSSION

On the first week of the experiment, the plasmid extraction of Gram-negative

bacteria was conducted. The working solution was prepared which is solution A,
solution B and solution C. The working concentration of solution was calculated
using M1V1 = M2V2 formula. The working concentration has to prepared carefully
with the exact volume for the plasmid extraction. If not, it will lead to inaccuracy of
precision methods. Since this experiment involves plenty of procedures which are
not only lengthy but they all require accurate quantity and timing, a slight
difference may be significant. Lengthy procedures call for longer handling of
materials, this actually allows an extended chance and time for contamination in
the DNA samples. Contamination of DNA may cause less DNA in the sample.
After the plasmid extraction was obtained it has been stored for one week, the
DNA samples are stored in a low temperature to prevent any spoilage to the DNA
samples.

Next, the agarose gel electrophoresis was conducted after one week of the
plasmid extraction was stored. Process starts with the preparation of gel
electrophoresis. Overall, the DNA fragments are placed in wells produced by the
combs, in an agarose gel medium in a buffer solution to maintain a constant pH.
The dye is also added together with the DNA samples not to bind with the DNA
but to move through the gel slightly faster than the DNA so that the current can
be turned off before all the samples run off the end. Electric current is passed
through the apparatus and DNA fragments move at different rates towards
positive anode because of negative charge on phosphate group in DNA.
Fragments move. Once, electrophoresis is complete, the plate is placed on a UV
transilluminator to visualize the DNA bands.

On the UV transilluminator, the image was not so clear. There are several
limitation have been identified in this experiment, it is the amount of DNA
available. The analysis of image of DNA bands after undergoing electrophoresis
showed that the bands were unclear and lesser than expected. This may be due
to the amount of DNA available in E. Coli culture is extremely small and
insignificant. The agar need to be in a good condition, due to some technical
errors and problems which arise due to lack of knowledge and thus, prolonging
the exact time for solidification of agar. As a result, the agar may be too hard in
order for the DNA fragments to move across. Thus, an appropriate time control
must be apply throughout the process. Besides, inaccurate use of micropipette
with teats may have caused smaller volume of DNA to be taken up and loaded
into the wells when preparing for electrophoresis.

CONCLUSION

From this experiment it is true that DNA from E. Coli culture can be extracted
and digested into fragments using restriction enzymes and separated by gel
electrophoresis. In the plasmid extraction of Gram-negative bacteria, a cell lysate
was prepared and the plasmid DNA from Gram-negative bacteria was eluded. In
agarose gel electrophoresis, the DNA fragment was seperated and observed on
UV transilluminator and the size of the DNA fragments was estimated.

REFERENCES

 https://learnsomescience.com
 www.kmbiology.com
 www.scribd.com