10 Agarose Gel Electrophoresis and Ligation

10.1 Agarose gel electrophoresis

Electrophoresis (from the Greek “electrophoron” meaning “to bear electrons”) is the motion of dispersed particles relative to a fluid under the influence of a spatially uniform electric field.

Agarose gel electrophoresis is a method of gel electrophoresis used in biochemistry, molecular biology, genetics, and clinical chemistry to separate a mixed population of macromolecules such as DNA or proteins in a matrix of agarose, one of the two main components of agar. The proteins may be separated by charge and/or size, and the DNA and RNA fragments by length. Biomolecules are separated by applying an electric field to move the charged molecules through an agarose matrix, and the biomolecules are separated by size in the agarose gel matrix.

Agarose gels are easy to cast and are particularly suitable for separating DNA of size range most often encountered in laboratories. The separated DNA is visualized using a stain, most commonly under UV light, and the DNA fragments can be extracted from the gel with relative ease. Most agarose gels used are made using 0.5–3% agarose which is dissolved in a suitable electrophoresis buffer.

Agarose gel is easy to cast, has relatively fewer charged groups, and is particularly suitable for separating DNA of size range most often encountered in laboratories, which accounts for the popularity of its use. The separated DNA may be viewed with stain, most commonly under UV light, and the DNA fragments can be extracted from the gel with relative ease. Most agarose gels used are between 0.7–2% dissolved in a suitable electrophoresis buffer.

10.2 Experimental Procedures

Take special care when pipetting very small volumes. Make sure only the soft stop of the pipet is used, when pulling up reagents even though it may feel like a very small movement. Also, look at the end of the pipet tip to be sure that the correct volume of reagent is in the tip. After adding the reagent to the tube, be sure that the pipet tip is empty. Never reuse a pipet tip.

Get the Erlenmeyer flask containing 1 g of agarose powder
Add 100 ml of 1× TAE (Tris base, acetic acid, EDTA) running buffer.
Add 10 µl of SybrGreen™ dye (10,000× stock solution).
Heat in the microwave at full power for 1 minute.
Swirl to make sure that all powder has dissolved, and the solution is clear.
Add the comb into the comb slot.
Pour the solution onto the gel tray in the gel box.
Label a microcentrifuge tube for each PCR sample.
Transfer 20 µl from each first-round PCR reaction tube into the labeled microcentrifuge tube. Add 4 µl of loading dye and stain into the tube. Pipette up and down to mix.
To assess the success of the nested PCR round, pipet 5 µl of each nested PCR into a microcentrifuge tube and mix it with 1 µl of 6× loading dye and stain. A smaller volume of the nested PCR is loaded because nested PCR is usually more efficient and produces very intense bands that can obscure bands in adjacent wells if samples are overloaded.
The gel will need about 30 minutes to solidify.
Start setting up the PCR product purification.

10.3 PCR Product Purification

10.4 Experimental Procedures

Label a PCR Kleen spin column and a cap-less collection tube with your initials.
Label a capped microcentrifuge tube with your initials, “purified PCR product,” and the plant name.
Resuspend the beads in the PCR Kleen spin column by vortexing. Return the beads to the bottom of the column with a sharp downward flick.
Snap the bottom off the spin column, remove the cap, and place the column in the cap-less collection tube. Discard cap and bottom of column.
Place the spin column, still in the cap-less collection tube, into the microcentrifuge. Make sure that your tube is placed in the rotor with another group’s or with a balance tube so that the microcentrifuge is balanced. Centrifuge columns at 735×g for 2 min. Do not use the top speed of the microcentrifuge for this step.
Move the spin column to the labeled microcentrifuge tube. Discard the flowthrough and the collection tube.
Pipet 30 µl of the nested PCR product onto the top of the column bed in the spin column, without disturbing the resin (or column bed). Save 5 µl of this original (not yet purified) yellow PCR sample and mix it with 1 µl of 6× loading dye and stain for gel electrophoresis.
Place the column in the labeled microcentrifuge tube into the microcentrifuge. It is best to orient the cap of the microcentrifuge tube downward, toward the center of the rotor, to minimize friction and damage to the cap during centrifugation. Centrifuge at 735×g for 2 min. Make sure that another group’s sample counterbalances the microcentrifuge.
Remove the spin column from the microcentrifuge tube and discard the column. Cap the microcentrifuge tube, which now contains the purified PCR product. Store at 4 °C for up to 2 weeks or at –20 °C long term.
Add 1 µl of 6× loading dye and stain into 5 µl of the purified sample.
Electrophorese 5 µl of this mix next to 5 µl of the not purified sample on a 1% agarose gel to visualize differences between the original and the purified sample.
Check if the agarose gel has solidified. If so, proceed to the next step or wait until the gel is ready.
Pour electrophoresis running buffer into the chamber until it just covers the gel by 1–2 mm.
Load 10 µl of the 500 bp molecular weight ruler into the first well.
Load 24 µl from each microcentrifuge tube containing an initial PCR with added loading dye. Load 6 µl from each tube containing a nested PCR with added loading dye into the wells of the gel according to your plan.
Connect your electrophoresis chamber to the power supply and turn on the power. Run the gel at 100 V for 30 min.
While the gel is running, start setting up the ligation reactions.

10.5 Ligation

In molecular biology, ligation is the joining of two nucleic acid fragments through the action of an enzyme. It is an essential laboratory procedure in the molecular cloning of DNA whereby DNA fragments are joined together to create recombinant DNA molecules, such as when a foreign DNA fragment is inserted into a plasmid. The ends of DNA fragments are joined together by the formation of phosphodiester bonds between the 3’-hydroxyl of one DNA terminus with the 5’-phosphoryl of another.

10.6 Experimental Procedures

Label a microcentrifuge tube with your initials and “L” for “Ligation.”
Briefly centrifuge the stock tubes containing the 2× ligation reaction buffer and proofreading polymerase in a microfuge to force contents to bottom of tubes.
Add 5.0 µl 2× ligation reaction buffer to the labeled microcentrifuge tube.
Add 1.0 µl purified PCR product
Add 2.5 µl sterile water
Add 0.5 µl proofreading polymerase
Close the cap and mix well. Centrifuge briefly in a microfuge to collect the contents at the bottom of the tube.
Incubate the tube at 70 °C for 5 min. 70 °C is the optimal temperature for the proofreading polymerase to blunt the PCR fragment.
Cool the tube on ice for 2 min. This re-condenses water vapor to maintain the reaction volume. Centrifuge the tube briefly to collect the contents at the bottom of the tube. Maintain the tube at room temperature.
Add 0.5 µl T4 DNA ligase.
Add 0.5 µl pJet1.2 blunted vector.
Close the cap and mix well.
Centrifuge briefly in a microcentrifuge to collect the contents at the bottom of the tube.
Incubate the tube at room temperature for 5–10 min.
Pipet 5 µl of the ligation reaction into a fresh microcentrifuge tube labeled with your initials and “t” for “transformation” and store it on ice. The laboratory techniccians will store the samples for us at -20 °C until we will perform the transformation during the next laboratory session.
Take the gel out of the electrophoresis box to visualize the DNA bands and acquire an image of your gel.

10.7 Review Questions

What is agarose gel electrophoresis?
What is the overall electrical charge of DNA?
What is a ligation?