Protocol: Agarose gel electrophoresis and gel staining (wear gloves, goggles, lab coat)
Instructor: Prepare a 1% agarose gel in advance (the day it will be used). You will only need 8 7
wells in the gel, so a mini gel apparatus with an 8-well comb will work fine. If you prepare two
gels, one can be used for students to practice loading gel wells. Safety: Wear gloves, goggles,
lab coat
1. Microwave already prepared 1% agarose in flask until boiling. [NOTE: The 1% agarose
solution was prepared by adding 100 ml 1X TAE and 1 g agarose in a 250 ml flask, swirling
the contents of the flask, and heating in a microwave until boiling.]
2. Allow the agarose to cool to ~50oC (agarose is hot, but it’s cool enough that you should be
able to hold the flask in your gloved hands).
3. Place a tray, 8-well comb, and buffer dams into an electrophoresis chamber. The comb
should be closest to the black (negative) end of the gel box.
4. Carefully pour ~50 ml agarose into the gel tray containing the comb which is sitting inside a
prepared gel electrophoresis chamber.
5. Allow the agarose to cool to room temperature. It will solidify during the cooling and will
become more opaque.
6. Once the gel has solidified, remove the buffer dams and pour 1 X TAE buffer over the top of
the gel and into both sides of the chamber until the buffer just covers the surface of the gel.
7. Carefully remove the comb by pulling it straight up (while holding the tray in place). This
forms the gel wells into which DNA will be loaded.
Students- loading the gel
1. Load each digested DNA sample (containing loading dye) into a separate well in the gel
using a P20 micropipettor. Hold the micropipettor vertically, and use a clean tip for each
sample. Insert the tip into the well when loading, but be careful to not poke through the
agarose at the bottom of the well.
Suggested loadings:
Lane 1- (10 µl) molecular weight marker DNA (with 1X loading dye)
Lane 2- (25 µl) control “evidence” DNA (CS) cut with EcoRI/PstI
Lane 3- (25 µl) DNA sample 1 (S1) cut with EcoRI/PstI
Lane 4- (25 µl) DNA sample 2 (S2) cut with EcoRI/PstI
Lane 5- (25 µl) DNA sample 3 (S3) cut with EcoRI/PstI
Lane 6- (25 µl) DNA sample 4 (S4) cut with EcoRI/PstI
Lane 7- (25 µl) DNA sample 5 (S5) cut with EcoRI/PstI
2. Once all samples are loaded, place the lid on the chamber, matching black with black, red
with red. Attach the leads to a compact power supply (again matching black with black, red
with red).
3. Turn on the power supply, 100 Volts and run the gel 30 minutes.
4. After 30 minutes, turn off the power supply. Remove the top from the apparatus.
5. Carefully remove the gel and stain it to visualize the DNA in the gel.
Staining/destaining the gel (wear gloves, goggles, lab coat)
DNA will be stained a deep blue color with Fast Blast Stain which binds the negatively charged
phosphate groups on DNA molecules.
1. Carefully slide the gel off the tray into a small (~8 cm X 12 cm) plastic box that contains 100
ml 100X Fast Blast DNA stain.
2. Stain the gels for 2 minutes with gentle agitation.
3. To destain the gel, carefully transfer the gel (using a pancake turner or spatula) into a large
plastic container (~18 cm X 18 cm) containing 500 ml warm (40–55°C) tap water and agitate
gently ~10 seconds.
4. Carfeully transfer the gel into a second large washing container with 500 ml warm tap water
and shake very gently ~5 minutes.
5. Carfeully transfer the gel into a third large washing container with 500 ml warm tap water
and shake very gently ~5 minutes.
6. Carfeully transfer the gel onto a large piece of saran wrap placed on a white surface to
visualize the gel.
7. Instructor: Carefully pour the used 100X Fast Blast DNA stain back into the original glass
bottle using a funnel (the stain can be saved and reused).
Heidi Sleister