Transcript LECTURE #20: Bacterial Transformation and Gel

DNA Technology:
BACTERIAL
TRANSFORMATION
Ms. Gaynor
Honors Genetics
What is Bacterial Transformation?
• Transformation
– “Naked” Plasmids (present in
environment) are taken up by certain
bacteria
– Viruses are NOT used in this method!
http://highered.mcgrawhill.com/sites/0072556781/s
tudent_view0/chapter13/ani
mation_quiz_1.html
Host E.coli cell is transformed 
bacteria takes in plasmid from environment
Bacterial Transformation
• Step 1 DNA Isolation
– Isolation of the “Gene of Interest” (foreign DNA)
• Step 2 Recombinant DNA
– Insertion of foreign DNA into bacterial plasmid
using restriction enzymes and DNA ligase
•
http://www.dnalc.org/resources/animations/transformation1.html
• Step 3 Transformation
– Insertion of recombinant DNA into
bacteria by making bacteria competent
(weaken)
• Use CaCl2 and heat shock techniques
How do you make Bacteria competent?
• Step 1: Add Calcium Chloride (CaCl2)
– CaCl2 is in a solution (creates Ca+2 and Cl- ions)
– DNA in plasmid is negatively charged due to
phosphate groups in the backbone
– Cell membrane of E. coli also is negatively
charged because phospholipids are made of
same phosphate groups (PO4-3)
– Ca+2 ions neutralize charges so plasmid can get
near (and inside) bacterial cell.
How do you make Bacteria competent?
• Step 2: Use Heat Shock
– Heat Shock is a process that uses
warm water (bath) and ice to help
get plasmid inside cell
• Add recombinant plasmid +
host cell + CaCl2 solution to
ice then heat then back on ice
– Heat = increases kinetic energy of
matter
• Molecules/atoms move faster
– Ice = decreases kinetic energy of
matter
• Molecules/atoms move slower
• http://www.dnalc.org/resources/animations/transfor
mation2.html
DNA Technology:
GEL
ELECTROHPHORESIS
Ms. Gaynor
Honors Genetics
DNA Gel Electrophoresis
DNA fingerprint
**Each band that you see is a collection of millions of
DNA molecules, all of the same length!!
Restriction Fragment Analysis
detects DNA differences that affect restriction sites
Gel electrophoresis
 Separates DNA restriction fragments of
different lengths
 Uses electrical current to separate DNA
based on size
 DNA has a negative charge.
 DNA moves towards the POSITIVE
electrode. Why?
 DNA molecules of SMALLER sizes move the
furthest through the gel.
http://www.sumanasinc.com/webc
ontent/animations/content/gelelect
rophoresis.html
Restriction
Fragment Analysis
Normal
Is useful for
comparing
two different
DNA
molecules,
such as two
alleles for a
gene

201 bp
175 bp
DdeI
-globin allele
DdeI
Large fragment
DdeI
Sickle-cell mutant
DdeI
-globin allele
Large fragment
376 bp
DdeI
DdeI
DdeI
(a) DdeI restriction sites in normal and sickle-cell alleles of
-globin gene.
Normal
allele
Sickle-cell
allele
Large
fragment
http://highered.mcgrawhill.com/sites/0072437316/student
_view0/chapter16/animations.html
#
376 bp
201 bp
175 bp
(b)
Electrophoresis of restriction fragments from normal and sickle-cell alleles.
Agarose Gel
Electrophoresis
1. Widely used technique for the analysis
of DNA (or RNA or proteins)
2. Routinely used (crime scenes,
maternity/paternity cases, etc)
3. Separates molecules based on their
rate of movement through a gel under
the influence of an electrical current
4. We will be using agarose gel (NOT
agar)
Purpose of Agarose Gel
Electrophoresis
 To separate a mixture of DNA
fragments by size using an electrical
charge
 The gel is a protein matrix (like a
sponge with holes; DNA travels
through “holes”)
• Polymerized agarose is porous,
allowing for the movement of DNA
Scanning Electron Micrograph
of Agarose Gel (1×1 µm)

How does gel electrophoresis
separate DNA fragments?
• Gel acts as a strainer to filter DNA by size
• DNA fragments are naturally negatively
charged due to the phosphate
backbone (PO4-3)
• DNA fragments of differing sizes will move
though the gel at differing rates
– larger fragments (more bases) = do
not travel as far from wells
– smaller fragments (less bases) =
travel farther from wells
Movement depends on
Charge
• DNA is negatively
charged (because
of phosphate
backbone)
• DNA will be
attracted to
positively
charged poles and
repelled from
negatively charged
ones
Movement Depends on Size
•Small DNA move faster than larger pieces DNA
•Gel electrophoresis separates DNA according to
size
•Power source supplies the electrical current
DNA
small
large
-
Power
+
Within an agarose gel, linear DNA migrate
inversely proportional to the log10 of their
molecular weight.
Restriction Enzymes and
Plasmid Mapping
Restriction Enzyme Digest 
different length pieces are made
Gel electrophoresis markers (called
standards or ladders) are used for size
identification of each DNA fragment
Each
well/column is a
“DNA
fingerprint”
Gel Electrophoresis Equipment
Power supply
Cover
Gel tank
Electrical leads

Casting tray
Gel combs
Making an Agarose
Gel
And Setting up
your Gel
Electrophoresis
Apparatus
Agarose:
D-galactose
3,6-anhydro L galactose
•Sweetened agarose gels
have been eaten in the
Far East since the 17th
century.
•Agarose was 1st used in
biology when Robert
Koch used it as a culture
medium for Tuberculosis
bacteria in 1882
Agarose is a linear
•Can be used to separate
polymer extracted DNA fragments > 300 bp
from seaweed.
An agarose gel is
prepared by
combining agarose
powder and a
buffer (ions + H2o)
solution into a
flask.
Buffer
Flask for boiling 
Agarose
A.
Agarose
Buffer Solution
Combine the agarose powder and buffer solution.
Use a flask that is several times larger than the
volume of buffer.
B.
Melting the Agarose
Agarose is insoluble at room temperature (left).
The agarose solution is boiled until clear (right).
Gently swirl the solution periodically when heating to
allow all the grains of agarose to dissolve.
***Be careful when boiling - the agarose solution may
become superheated and may boil violently if it has
been heated too long in a microwave oven.
C.
Gel casting tray & combs
Cast (make) the
gel using this
tray and comb
C.
Preparing the Casting Tray
COMBS CREATE
WELLS!!!
Seal the edges of the casting tray and put in one comb with 13 teeth.
Place the casting tray on a level surface. None of the gel combs
should be touching the surface of the casting tray.
D.
Pouring the gel
Allow the agarose solution to cool slightly (~60ºC)
and then carefully pour the melted agarose solution
into the casting tray. Avoid air bubble, why?
D.
Make sure that the gel combs are
submerged in the melted agarose
solution but not touching the bottom.
E.
When cooled, agarose polymerizes, forming a
flexible gel. It appears cloudy in color when
completely cooled (~20 minutes). Carefully
remove comb (be very, very careful…don’t
remove at an angle!).
Place the gel in the electrophoresis
chamber.
DNA
buffer 

wells
Cathode
(negative end)
BLACK WIRE!



Anode
(positive end)
RED WIRE!
Add enough buffer to cover the gel to a depth of at
least 1 mm. Make sure each well is filled with buffer.
Buffer allows electrical current to FLOW
through chamber!
REVIEW…Loading and
Running the gel
• Molten agarose is poured into a casting tray
and a comb is placed inside the casting tray.
• After the agarose solidifies, the comb is
removed leaving wells where the DNA will be
loaded.
• DNA samples are mixed with tracking dye
which contains glycerol (to weigh down the
DNA into the well) and acts as a mobile dye
so that you can visualize migration
– this is why the DNA “falls” into the wells and you
can SEE it move through the gel!!!
• A buffer containing ions (to conduct an
electric current) is placed in the chamber
around the gel after
Sample Preparation
Samples of DNA need to be mixed with tracking dye.
•Allows DNA samples to be seen in the gel
•Increases the density of samples, causing them
to sink into the gel wells.
Loading Dye:  FUNCTIONS:
 Bromophenol Blue (for color)
 Glycerol (for weight)
Loading the Gel
Carefully place the micropipette tip over a well and
gently expel the sample. The sample should sink
into the well NOT float in the buffer. Be careful
not to puncture the gel with the pipette tip.
Running the Gel
Place the cover on the electrophoresis chamber and
connect the electrical leads. Be sure the leads are
attached correctly - DNA migrates toward the anode
(red). When the power is turned on, bubbles should
form on the electrodes in the electrophoresis chamber.
 wells
Cathode
(-)
End
DNA
(-)

Migration
 Bromophenol Blue
Gel
Anode
(+)
End
After the current is applied, make sure the Gel is
running in the correct direction. Bromophenol blue
will run in the same direction as the DNA.
Staining the Gel
• Ethidium bromide binds to DNA and fluoresces under UV light,
allowing the visualization of DNA on a Gel.
YOU ARE USING A QUICK DNA STAIN!!!
• Ethidium bromide can be added to the gel and/or running buffer
before the gel is run or the gel can be stained after it has run.
***CAUTION! Ethidium bromide is a powerful mutagen and is
moderately toxic. Gloves should be worn at all times.
Staining the Gel
• Place the gel in the staining tray containing warm diluted
stain.
• Allow the gel to stain for 25-30 minutes.
• To remove excess stain, allow the gel to destain in water.
• Replace water several times for efficient destain.
Staining the Gel
• Place the gel in the staining tray containing warm
diluted stain.
• Allow the gel to stain for 15-20 minutes.
• To remove excess stain, allow the gel to destain in
water.
• Replace water several times for efficient destain.
Methylene blue requires an ultraviolet light source to visualize
Visualizing the DNA
DNA ladder/Size standard

1
2
3
4
5
6
7
DNA ladder

8
wells
 5,000 bp
 2,000
 1,650
 1,000
 850
 650
 500
 400
 300
 200
 100
DNA:
+
-
-
+
-
+
+
-
Samples # 1, 4, 6 & 7 were positive for DNA samples taken
from the crime and compared to suspect
Visualizing the DNA (Actual Image)
wells
DNA ladder

DNA
+ - - - - + + - - + - +
 2,000 bp
 1,500
 1,000
 750
 500
 250
Samples # 1, 6, 7, 10 & 12 were positive for our suspect and
crime scene samples
March 12, 2006
Movement of DNA
fragments
in agarose gels
• There is a linear relationship
between the migration rate of a
given DNA fragment and the
logarithm of its size (in basepairs).
• Larger molecules move more slowly
through the gel because of more
friction
Semilog paper
Fragment Length (bp)
GRAPH THE LADDER/STANDARD…then
make a best fit line or curve!
Distance migrated (mm)
Fragment
Length (bp)
x bp
Distance migrated (mm)