*
Practical #2: Extraction of genomic DNA from E.Coli
Practical #3: Agarose Gel Electrophoresis
Bertrand Ong
Chan JianPeng
Salanne Lee
*Introduction
*Materials & Procedure
*Results & Discussion
*Conclusion
*Q&A
*
* DNA (Deoxyribonucleic acid)
* Contains genetic makeup of all living organisms
* Bases: Adenine (A), Thymine (T), Guanine (G), Cytosine (C)
* Base + Phosphate Group + Deoxyribose Sugar = Nucleotide
Nucleotide!
*
*Escherichia Coli, or E.Coli are
prokaryotic cells (no nucleus)
*Circular DNA, also known as
PLASMIDS  found in nucleoid
*Many strains of E. Coli exists
*
*Extract genomic DNA from bacterial
cultures
*Determine the yield and quality of DNA
using Nanodrop
*Separate genomic DNA using agarose
gel electrophoresis
*Learn how to capture a gel image using
a UV transilluminator
*
usually used to determine the
concentration of DNA and RNA
in a mixture.
* Nanodrop
(spectrophotometer) expose a
sample to ultraviolet light
* photo-detector measures the
light that passes through the
sample
* The more light absorbed by
the sample, the higher the
nucleic acid concentration.
*
* Agarose gel electrophoresis is a
frequently used technique in
molecular biology to separate
DNA or RNA fragments by size.
* determine the size of PCR
products and to isolate DNA
fragments after restriction
enzyme digestion
PRACTICAL #3
PRACTICAL #2
* Nucleic acid quantification is
* E.coli bacterial cultures
* Qiagen DNeasy blood and
*
tissue kit
* Microcentrifuge tube
* Microcentrifuge
* Agarose gel powder
* Conical flask
* Gel red, DNA ladder, loading
dye, TBE buffer
* Gel tanks
* Power pack
* Extracted E.coli DNA
* Bio-Rad Doc imaging system
1. The bacterial cells are spun down
for 10 min at 4000 rpm.
2. Pellet is suspended in 180µl Buffer
ATL. Cell suspension is then pipetted
into a microcentrifuge tube.
3. 200µl Buffer AL is then added to
the sample, mixed and incubated at
56⁰C for 10min.
4. 200µl ethanol (96 - 100%) is then
added to the sample and thoroughly
mixed.
*
5. The mixture is pipetted into the DNeasy
Mini spin column and centrifuged at 8000
rpm for 1 min. The filtrate is discarded.
6. The DNeasy Mini spin column is placed back
into the collection tube. 500µl Buffer AW1 is
added and centrifuged at 8000 rpm for 1 min.
The filtrate is discarded.
7.Step 6 is repeated but this time with
Buffer AW2 and centrifuging of the mixture
occurs at 14000 rpm.
8. The DNeasy Mini Spin column is then placed into a
microcentrifuge tube and 200µl Buffer AE is pipetted
directly onto the DNeasy membrane. The mixture is
incubated at room temperature for 1 min and
centrifuged for 1 min at 8000 rpm to elute.
*
1.
1.2g of agarose is
weighed.
2. 100ml of 1X Tris BorateEDTA (TBE) buffer is added.
4. 5µl of gel red is
added into the
molten agarose
*
5. It is then poured
into a gel caster (preprepared with a well
comb.
6. The well comb is gently removed
from the gel once the gel has set.
7. C1X TBE buffer is then
poured into the gel tank until
the gel is submerged
*
8. 2µl of loading dye is then
mixed with 10µl of extracted DNA
on parafilm and loaded into the
wells. 5µl of 1 kbp DNA ladder is
loaded into 1 of the wells
9. Electrophoresis is then run at
90V for 40 min.
*
10. The DNA bands are then visualised
using the UV transilluminator (Bio-Rad Gel
Doc imaging system)
We have collected our results
The following value is the ratio of
A260/A280 ratio change
• Value:1.90 (A260/A280)
• Value: 0.99 (A260/A230)
• DNA sample
PURE ! (within range of 1.8)
• Nucleic Acid
Contain Contaminants (out of range)
*
• Makes use of UV light to
illuminate the resultant DNA
fragments
• Prior to Illumination,
fluorescent dye is added to
the gel.
e.g. Ethidium bromide, gel red
UV transilluminator with an image analysis software
*
• Consist of illuminated DNA
fragments (In white)
• With white markings of
different lengths
Our Result
*
• Usually includes a DNA
Marker
* The further the ‘white pattern’ travels, the smaller the DNA
molecule.
* Allows differentiating of the molecular mass of DNA
molecules
*
* Genomic DNA was extracted in the 2nd practical
and the quality of the DNA was experimentally
determined to be pure (except for the purity
of nucleic acids).
* In the 3rd practical, agarose gel electrophoresis
was conducted and the visualizing image was
collected.
*
*
P.S. PLEASE BE NICE 
*
1. Microcentrifuge tube [online]. USA: Sorenson, BioScience, Inc. Availble
from: http://www.sorbio.com/content/65-mct-nat-s_1.jpg[Accessed 6 July
2011]
2. Nucleotide [online]. USA: Sciencey Splurge. Availble from:
http://scienceysplurge.files.wordpress.com/2010/10/nucleotide.jpg
[Accessed 6 July 2011]
3. E.Coli [online]. USA: Wordpress. Available from:
http://2012patriot.files.wordpress.com/2011/06/ecoli2.jpg [Accessed 6
July 2011]
4.E.Coli: Black & White [online]. USA: Wikipedia. Available from:
http://upload.wikimedia.org/wikipedia/commons/thumb/3/32/Escherichia
Coli_NIAID.jpg/250px-EscherichiaColi_NIAID.jpg [Accessed 11 July 2011]
5. DNA Electrophoresis Size Standards [online]. USA: Bio-Rad. Available
from: http://www.biorad.com/webroot/web/images/lsr/products/imaging_bioinformatics/produ
ct_overlay_content/global/lsr_geldoc_sizestds.gif [Accessed 11 July 2011]
*