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.mcgraw-hill.com/sites/0072556781/student_view0/chapter13/animation_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/

transformation2.html

DNA Technology: GEL ELECTROHPHORESIS

Ms. Gaynor

Honors Genetics

DNA Gel Electrophoresis

**Each band that you see is a collection of millions of DNA molecules, all of the same length!!

Restriction Fragment Analysisdetects DNA differences that affect restriction sites

DNA fingerprint

Gel electrophoresisSeparates DNA restriction fragments of

different lengthsUses electrical current to separate DNA

based on sizeDNA 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/webcontent/animations/content/gelelectrophoresis.html

Restriction Fragment Analysis

Is useful for comparing two different DNA molecules, such as two alleles for a gene

http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter16/animations.html#

Normal -globin allele

Sickle-cell mutant -globin allele

175 bp 201 bp Large fragment

DdeI DdeI DdeI DdeI

DdeI DdeI DdeI

376 bp Large fragment

DdeI restriction sites in normal and sickle-cell alleles of -globin gene.

Electrophoresis of restriction fragments from normal and sickle-cell alleles.

Normalallele

Sickle-cellallele

Largefragment

201 bp175 bp

376 bp

(a)

(b)

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)

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”)

Scanning Electron Micrograph

of Agarose Gel (1×1 µm)

• Polymerized agarose is porous, allowing for the movement of DNA

Purpose of Agarose Gel Electrophoresis

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

+-

Power

DNA

•Small DNA move faster than larger pieces DNA•Gel electrophoresis separates DNA according to size•Power source supplies the electrical current

smalllarge

Within an agarose gel, linear DNA migrate inversely proportional to the log10 of their

molecular weight.

Movement Depends on Size

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”

Casting tray

Gel combs

Power supply

Gel tank Cover

Electrical leads

Gel Electrophoresis Equipment

Making an Agarose GelAnd Setting up your

Gel Electrophoresis

Apparatus

Agarose:

Agarose is a linear polymer extracted

from seaweed.

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•Can be used to separate DNA fragments > 300 bp

An agarose gel is prepared by combining agarose powder and a buffer (ions + H2o) solution into a flask.

Agarose

Buffer

Flask for boiling

Agarose Buffer Solution

Combine the agarose powder and buffer solution. Use a flask that is several times larger than the

volume of buffer.

A.

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.

Melting the AgaroseB.

Gel casting tray & combs

Cast (make) the gel using this tray and comb

C.

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.

Preparing the Casting TrayC.

COMBS CREATE WELLS!!!

Allow the agarose solution to cool slightly (~60ºC) and then carefully pour the melted agarose solution

into the casting tray. Avoid air bubble, why?

Pouring the gelD.

Make sure that the gel combs are submerged in the melted agarose

solution but not touching the bottom.

D.

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!).

E.

Place the gel in the electrophoresis chamber.

buffer

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!

Cathode(negative end)BLACK WIRE!

Anode(positive end)RED WIRE!

wells

DNA

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

Loading Dye: FUNCTIONS: Bromophenol Blue (for color) Glycerol (for weight)

Sample PreparationSamples 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 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.

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.

Running the Gel

w

ells

Bromophenol Blue

Cathode(-)

End

Anode(+)End

Gel

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.

DNA(-)

Migration

Staining the Gel

***CAUTION! Ethidium bromide is a powerful mutagen and is moderately toxic. Gloves should be worn at all times.

• 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.

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

100 200 300

1,650 1,000

500

850 650

400

5,000 bp 2,000

DNA ladder

DNA ladder/Size standard

DNA:

1 2 3 4 5 6 7 8

wells

+ - - + - + + -Samples # 1, 4, 6 & 7 were positive for DNA samples taken from the crime and compared to suspect

Visualizing the DNA (Actual Image)

250

1,500 1,000

500 750

2,000 bp

DNA ladder

DNA

wells

+ - - - - + + - - + - +

March 12, 2006

Samples # 1, 6, 7, 10 & 12 were positive for our suspect and crime scene samples

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

Distance migrated (mm)

Fra

gmen

t Le

ngth

(bp

)

GRAPH THE LADDER/STANDARD…then make a best fit line or curve!

Distance migrated (mm)

Fra

gmen

t Le

ngth

(bp

)

x bp