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PRACTICAL 2

Materials:

70 % Ethanol (cold, -20°C)

TE buffer (10mM Tris-CI, 1mM EDTA, pH 7.5)

Loading buffer, 5X

Parafilm

Certified molecular biology Agarose

Ethidium Bromide (EtBr) solution (10mgm1), Scoop, Stained and Destained tray

1X Trio Acetic acid EDTA (TAE)

Marker 1kb ladder

Sterilized 1.5 ml microcentrifuge tubes

Sterilized tips (1m1, 200p1 , 10p1)

Steriled microcentrifuge tubes

Microcentrifuge racks

Sterilized distilled water

Ice box with ice

Gloves

Equipment:

Micropipette set

Bench top centrifugation

Speedvac + vacuum pump

Electrophoresis set + comb


Power supply

Freezer

Vortex

Imager system

1.0 LAB PROCEDURE:

1.1 ETHANOL PRECIPITATION OF DNA (CONTINUE)

We centrifuged at 12 000-15 000 rpm for 10 min at room temperature. We carefully removed
the liquid from the tube and add 1m1 of 70, ethanol (- 20°C) followed by vortex. Then, we
centrifuged again for 2 min and aspirated the supernatant. The supernatant was removed as
much as we can and followed by drying in a vacuum for 5-10 min. We dissolved the precipitate
in TE buffer with the suitable volume.

1.2 DNA ANALYSIS (ELECTROPHORESIS)


Charged macromolecules can be separated according to size and charge by a process called
electrophoresis. The material to be tested is placed at one end of a gel matrix. Polyacrylamide
gel or agarose is used to separate proteins and small pieces of DNA and RNA. Agarose is the
usual matrix for separation of nucleic acids. A buffer (salt solution) is used to conduct electric
current through the matrix. When electric current is applied, the molecules start to move
Molecules with different charges and sizes will migrate at different rates.

1.2.1 Gel Preparation


We determined the amount of agarose (grams) required to make a desired agarose gel
concentration and volume 1 % agarose (40 ml) = 0.4 g agarose + 40 ml 1X TAE buffer.
We added the agarose to the conical flask containing buffer. We heated in a microwave or
use a magnetic hot plate until the agarose was dissolved and swirled to mix. We left it to
cool for 5 minutes down to about 55 °C before pouring. (If hot solution had been poured
to the tank, the gel will be crooked).

1.2.2 Casting Agarose Gel Slab


We slided the gel casting gates into the slots at the opposite ends of the gel stage. We
inserted the comb and make sure the sample wells were near the cathode/ -ve pole (black).
DNA sample migrated toward the anode/ +ve pole (red) during electrophoresis. We poured
melted agarose onto the casting tray until the agar surrounds the comb. Usually the gel
thickness is around 5mm. We pushed any bubbles away to the side using a disposable tip.
The agar should have a flat surface. Then, we let the gel harden for about 20 min.

1.2.3 Loading the Sample and Electrophoresis


After the gel was being solidified (20 min), we carefully removed the combs to make sure
that the gel and the wells did not tear. We poured the buffer until the buffer just covers
the agarose gel and not exceed than 1mm. Make sure that the bubbles are not trapped in
the well. We prepared the DNA samples by mixing each DNA solution with loading
buffer. The volume for each sample should not exceed than the volume of the well. On a
piece of Parafilm, we put a few drops (1 ul each) of loading buffer or tracking dye (Ratio
of sample: loading dye; 5:1). To the first drop, we added the sample (5 ul of DNA
plasmid or 10 ul of PCR product) and mix well before loading it in the well of the gel. By
using micropipette, we loaded the sample into the second well of the gel. Loaded the first
well with 5 ul of DNA marker (1 kb DNA ladder). Avoid from mixing the sample from
different well. Then, we switched on the electrophoresis. Set for the volt: usually at 1-
10V per cm gel. Ran at 100 volts for approximately 30 minutes, or until the blue tracking
dye migrated to a little more than halfway down the length of the gel. We monitored the
progress of the gel from time to time by referring to bromophenol blue stained
movement. We stopped the gel by turning off power supply after 30 minutes or when the
bromophenol blue reached 3/4 the length of the gel. We removed the gel from tank and
stained with 0.5ug/ml ethidium bromide solution for 10-30 min. Ethidium bromide is a
known mutagen and a potential carcinogen. We wpre latex glove when handling the
reagent. We rinsed the gel with distilled water and photographed it on UV
transilluminator. We avoided our eyes from exposed to any of UV light.

1.2.4 Ethidium Bromide Staining Procedure


We placed the gel into the appropriate volume of 0.5 ug/ml ethidium bromide (Etbr)
stained for 15-30 minutes. Used enough staining solution to cover the entire gel.
CAUTION: Ethidium bromide is a suspected carcinogen and should be handled with
extreme care. Always wear gloves, eye glasses, and a laboratory coat. We disposed of
used Etbr solutions and gels appropriately. We destained the gel for 10-30 minutes in
dH20 using the same volume used for staining. NOTE: Ethidium Bromide can be
removed from the DNA with extended destaining. This will cause lower sensitivity of
detection. However, insufficient destaining will create higher background fluorescence.
We rinse the gel briefly with dH20 to remove any residual staining solution. We placed
the gel on a UV transilluminator for nucleic acid visualization and analysis. DNA-Etbr
complexes may be illuminated with UV light.