A method used for the separation of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or protein molecules using an electric current applied to a gel matrix.
Forensics, molecular biology experiment, genetics, microbiology and biochemistry
This experiment contains toxic chemicals as well as extreme sterilization condition. Please refer to the Safety section for careful handling of chemicals.
Gel refers to a crosslinked polymer that can contain and separate a target molecule due to its pores inside. It is usually composed of different concentrations of acrylamide and a cross-linker, producing different mesh networks of polyacrylamide or agrose.
Electrophoresis refers to the electromotive force (EMF) that is required to move the molecules through the gel. Nucleic acids are negatively charged but placing them in a well that is on the negative side of the EMF will make them migrate towards the positive side of the gel.
Based on the size of the molecule, they will migrate at different rates. The lighter the molecule, the faster the molecule travels.
And in the case of nucleic acid, since it is one dimentional linear structure, the longer, or more base pairs one nucleic acid molecule contains, the heavier the molecule is. By letting different sample traveling over the same period of the time, they will stop at different locations. The distance travelled by the molecule is then inversely proportional to the mass of the molecule and can be used to determine what the unknown sample is by comparing its location to a unknown sample.
- Gelbox apparatus with plates
- Plastic comb
- TBE and TAE
- Power source
- Possibly centrifuge
- Ethidium Bromide or SyBR Green
- Dark room or alumium foil
- UV light in Chamber
- Material used in this experiment is highly toxic and can not be easily accessed at home. Therefore, recommending high school students to ask permission for conducting the procedures either at an accredited university, their school if the apparatus are available, or at community college. Email the professors and teachers before the student go. Please refer to the Safety section.
- Make cross-linked polymer gel (agarose) with a row of wells, or holes, on top by inserting comb by the end.
- Attention: To ensure the tube will not be broken by dropping the crystal, the iodine solid is slipped along the side to the bottom of the tube while holding the tube in an angle.
- Emerge the gel in Tris/Borate/EDTA (TBE) buffer.
- Place the gel spans the positive and negative end of a power supply, and place the wells on the negative end.
- Mix the solution containing different samples, either DNA, RNA or protein, with negatively charged blue tracking dye (coomassie).
- Centrifuge the sample to get solution go to the bottom of the tube.
- Load sample and a ladder, which contains DNA with different sizes, with equal proportion to each of the well using a micropipette.
- Once the iodine sublimates throughout the tube, the secretions or unsaturated fatty acids in fingerprint residue will be transferred onto a porous surface, a.k.a, a white paper.Cut the big paper into slices to fit into the tube.
- Attention: Make sure no markings are presented on the paper and the fingerprints will be clearly recognized on the paper.
- Open the power supply, the DNA molecule with the blue dye starts to migrate.
- Wait until the front of the blue dye reaches the bottom of the gel, and turn off the power supply.
- Stain the gel in TBE solution mixed with small amount of ethidium brocmide or SYBR Green in a container, and slightly shakes the container for about 15 minutes under condition that is not exposed to light.
- Visualize the gel under ultraviolet (UV) light, and take picture of the gel.
If automated softwares are available in conjunction with the UV camera, use the software to determine the size of the molecule by highlighting the band presented on the photo. The number of basepairs contained in each sample will be calculated and reported.
If automated softwares are not available, and accurate analysis is not required, the relative size of the molecule can be obtained by comparing the location of the band in the sample with the location of bands in the ladder. If the location is in the middle of two ladder bands, the number of basepairs in the sample band is then in the middle of those two ladder bands.