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BIO 103 GENERAL BIOLOGY

PART I: Agarose Gel Electrophoresis

Agarose Gel Electrophoresis


The standard method used to separate and identify DNA fragments The electrophoretic migration rate of DNA through agarose gels is dependent upon four main parameters.
The molecular size of the DNA The agarose concentration The confirmation of the DNA The applied current

Main Components of the Agarose Gel Electrophoresis System


Gel Electrophoresis Tanks and Power supply

Buffers
Several different electrophoresis buffers are available (Tris-acetate (TAE), Tris-phosphate(TPE), Tris-borate (TBE)

Procedure
A. Casting an agarose gel:
Make 50ml of a 0.8% (w/v) solution of agarose in 1X TAE buffer. Heat the mixture until boiling using the heater. When the agarose is totally dissolved, wait for cooling for a few minutes at room temperature. Add ethidium bromide (EtBr) before pouring the gel. Put the wedges and the comb in place.

Pour the agarose solution into the casting tray.


Leave the gel to cool and solidify.

Ethidium bromide : is incorporated into the gel When exposed to ultraviolet light, it will flouresce. Ethidium bromide is a very strong mutagen and a possibly carcinogen. nucleic acid stain: Ethidium binds by inserting itself between the stacked bases in double-stranded DNA.

B. Preparing the samples: Prepare the DNA samples by adding 10 l of loading dye (bromophenol blue) to 2 l of DNA sample.
Loading dye is bromophenol blue and contains 50% glycerol, adding the dye will increase the density so the samples falls into the well of the gel. Loading dye also provides a visible marker to monitor the electrophoresis process.

Also prepare a molecular size (already prepared by the assistants)

Loading and running the gel: Remove the wedges and the comb very carefully. Add TBE buffer just over the gel. Carefully pipette each sample into a well.

When all samples are loaded, connect the leads from the power supply to the gel. Make sure that the gel is oriented correctly!!!!
Black: negative Red: positive DNA is a negatively charged molecule, so it runs from black to red.

Set the voltage and turn on the power. Run the gel until the tracking dye is approx. the way of the gel.

Photography:
Place the gel on the transilluminator. Turn on the machine. UV light is hibhly carcinogen so make sure that the lid is closed!!! Observe.

Each band corresponds to 200 base pairs.

500 400 300 200

100 ~80

1000 bp ladder

DNA samples

BIO 103 GENERAL BIOLOGY

PART II: Quantification of DNA

Determine the concentration and purity of the DNA sample :


1) Prepare 1/100 dilution of your plasmid DNA in 1 ml (1000 l) TE buffer 2) Transfer this solution to quartz cuvette by micropipette 3) Put 1 ml TE buffer in another quartz cuvette (blank solution) 4) Adjust absorbance of your blank solution to zero at 260 nm. 5) Measure absorbance of the diluted plasmid DNA at 260 nm. 6) Repeat steps 4 and 5 to measure OD at 280 nm. 7) If your measured OD values are more than 2, make more diluted DNA solution and repeat your measurements at 260 and 280 nm.

Calculate the DNA concentration : 1 OD260: 50 g/ml of DNA


DNA Concentration (mg/ml) = A260 x 50 x dilution factor A260 = optical density (OD) reading at 260 nm

Calculate the purity: OD260/OD280 : 1.8 for pure DNA Since proteins tends to absorb at 280nm, less than the values above means protein contamination