Agarose gel electrophoresis
Reagents
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Agarose powder
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TBE buffer
-
DNA loading dye
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DNA ladder/marker (e.g., 1 kb
ladder)
-
Gel stain (e.g. SYBR Safe,
Ethidium bromide)
Equipment
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Gel casting tray and comb
-
Gel electrophoresis chamber
-
Microwave or hot plate
-
UV or blue-light
transilluminator
Procedure
1. Prepare
Agarose Gel
-
Weigh 0.3 g of agarose per 30
mL (for 1 % gel).
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Mix with 50 mL TBE buffer in a flask.
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Heat in microwave until fully
dissolved (30–90 sec, swirling intermittently).
-
Cool to ~60°C. Add gel stain
(e.g., 3 µL of SYBR Safe or 2 µL EtBr).
-
Pour into casting tray with
comb inserted.
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Let solidify at room temp
(20–30 min).
2. Prepare
Samples
-
Mix DNA sample with loading dye
(usually 1:5 ratio, e.g., 2 µL dye + 10 µL DNA).
-
Thaw and prepare DNA ladder as
reference.
3. Run the Gel
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Place gel into electrophoresis
chamber and cover with 1× TAE/TBE buffer.
-
Carefully load samples and
ladder into wells.
-
Connect the electrodes: Black =
negative, Red = positive (DNA runs to Positive).
-
Run at 90 V, 400 mA for 30 minutes (until dye reaches
2/3 of the gel length).
4. Visualize
DNA
-
After the run, place gel on a
UV or blue-light transilluminator.
-
Capture image and document band
sizes.
Notes
-
Ethidium bromide is mutagenic.
-
Always wear gloves and
protective eyewear when handling gel stains and UV light.