Gel Extraction
Used to extract and purify DNA fragments
from agarose gel using the Wizard® SV system, yielding high-quality DNA
suitable for restriction digestion, ligation, or transformation.
Reagents and
Equipment Required
-
Wizard® SV Gel and PCR Clean-Up
Kit (Promega)
o
Membrane-based spin columns
o
Binding Buffer (includes
guanidine salt)
o
Wash Buffer (with ethanol,
labeled “Wash Buffer”)
o
Elution Buffer (or
nuclease-free water)
-
Microcentrifuge tubes (1.5 mL
or 2 mL)
-
UV transilluminator or blue
light viewer
-
Scalpel or clean razor blade
-
Microcentrifuge (≥13,000 rpm)
-
Heating block or water bath
(optional, for better elution)
Protocol Steps
1. Excise and
weigh DNA Band
-
Use a clean scalpel to cut out
the DNA band from the agarose gel.
-
Place the gel slice in a 1.5 mL
tube.
-
Record the weight.
2. Dissolve
Gel Slice
-
Add 10 µL of Membrane Binding
Solution per 10 mg of gel slice.
-
Incubate at 50–65°C until the
gel is fully dissolved.
3. Bind DNA to
Column
-
Insert SV spin column into a
collection tube.
-
Transfer the dissolved gel
solution to the spin column.
-
Centrifuge at 16,000 × g for 1
minute.
-
Discard the flow-through and
reinsert the column into the same tube.
4. Wash Column
-
Add 700 µL of Wash Buffer and centrifuge
for 1 minute at 16000 g.
-
Discard the flow-through, add
700 µL of Wash Buffer and centrifuge for 5 minutes at 16000 g.
-
Discard the flow-through and centrifuge
again for 1 minute to remove residual ethanol.
5. Elute DNA
-
Transfer column to a new, clean
1.5 mL tube.
-
Add 30 µL of Nuclease-Free
Water or Elution Buffer directly to the center of the membrane.
-
Incubate for 1 minute at room
temperature.
-
Centrifuge at 16000g for 1
minute to elute DNA.