Genetic Engineering

PRACTICAL Extraction of Genomic from Bacterial

Objectives
1. To extract genomic material of Salmonella through the conventional method according
to Wilson technique.
2. To familiarize student on how to do aseptic lab work.

Methodology
A singe colony grown overnight was transferred into a new 1.5ml eppendorf tube. Cell was
pelleted by microcentrifuge at 13000rpm for 1 minute. Cell pellet obtained was resuspended
in 700µl of TE buffer (pH 8) and 25% SDS and the mixture was vortexed. The samples were
incubated in water bath at 60°C for 15 minutes until the solution become clear. The samples
were inverted every 5 minutes during the incubation. Then, 800µl of chloroform
isoamylalcohol was added and mixed gently.

The resultant cell lysate solution was centrifuged at 13000rpm for 1 minute. 200µl of upper
aqueous layer was carefully transferred into a new sterile 1.5ml eppendorf tube. An equal
volume of 3M sodium acetate was added and mixed gently. A double volume of cold
isopropanol was added and centrifuged at 13000rpm for 10 minutes. The resultant
supernatant was discarded. The pellet was washed twice with 500µl of 70% cold ethanol and
spun at 12000rpm for 5 minutes. The ethanol solution was discarded. The pellet was dried at
room temperature and resuspended in 50µl of sterile distilled water. The DNA solution was
stored at -4°C until further use.

Then 15µl of genomic solution was loaded into the well in 0.8% agarose gel. The gel was run
at 75V at 5 minutes and 70V at 40 minutes. The agarose gel was stained in the ethidium
bromide and visualize under UV light.

Question
1. Explain why we use the PCI solution.
PCI solution was used for purification of DNA and RNA. Proteins and restriction enzymes
are removed by phenol and chloroform in disrupting protein secondary structure causing
proteins to denature and precipitate from solution. Although each of these solvents is
capable of performing this function alone, the two materials together remove proteins
from solution much more effectively. The isoamylalcohol reduces foam, which is a
problem with phenol-chloroform.

2. What are the functions of isopropanol and ethanol in plasmid isolation?

Isopropanol is used to precipitate the DNA, but does not completely remove salts.
Ethanol was added to ensure that. The ethanol-washed pellet contains the plasmid DNA,
together with some RNA.

3. We stained the agarose gel in the ethidium bromide. Explain how the band could be
florescence colour.
Ethidium bromide is an intercalating1 agent which is the most common dye used to make
DNA or RNA bands visible for agarose gel electrophoresis. When exposed to ultraviolet
light, it will fluoresce with an orange colour, intensifying almost 20-fold after binding to
DNA.

1
reversible inclusion of a molecule (or group) between two other molecules (or groups).

1
 Genetic Engineering

The hydrophobic environment found between the base pairs is believed to be responsible
for the fluorescence. By moving into this hydrophobic environment and away from the
solvent, the ethidium cation is forced to shed any water molecules that were associated
with it. As water is a highly efficient fluorescent quencher, the removal of these water
molecules allows the ethidium to fluoresce.

Conclusion
Through this experiment, we become familiar with the aseptic technique to extract genomic
material of Salmonella through the conventional method according to Wilson technique.

References
http://www.interchim.com/interchim/bio/cat_bio/pdf/Genomics_nucleic_acid_Biochemical
s.pdf (130908)

http://en.wikipedia.org/wiki/Ethidium_bromide (130908)

2
 Genetic Engineering

DNA Extraction Group 1

Lab Genetic Engineering
110908 (Thursday)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

0.8% of agarose gel

Lane 1 : Marker (7µl 100bp plus + 6µl 6x loading dye)
Each 20µl of sample:
Lane 2 : Preeya
Lane 3 : AT
Lane 4 : FN
Lane 5 : ATC
Lane 6 : 40A
Lane 7 : ATC 85
Lane 8 : ATC WAN
Lane 9 : ATC RU&SYIKIN
Lane 10:ATC EKA
Lane 11:S40AM
Lane 12:S40LY
Lane 13:S40LS
Lane 14:S40LUVA
Lane 15:S40
Lane 16:S40
Lane 17:S40NG21