DNA sequences can be enzymatically or chemically degraded to produce a series of sets of DNA fragments that can be resolved by thin-layer denaturing polyacrylamide gel electrophoresis. A typical denaturing polyacrylamide gel electrophoresis is capable of resolving DNA sequences encoding 15 to 400 nucleotides in length. This experiment is from the next volume of the Laboratory Guide to Molecular Cloning (Third Edition) by J. Sambrook D.W. Russell.
Operation method
Sampling and DNA sequencing
Materials and Instruments
Buffers and solutions 0.5XTBE and or 1XTBE gels Nucleic acids and oligonucleotides Move makings For more product details, please visit Aladdin Scientific website.
Automatic microfuge Bull's-eye clamps Gel temperature monitoring strips Pasteur pipettes Plates with plastic coating Regulated power supply Dissecting blades Shark-tooth combs Syringes 85°C water baths and 100°C ovens
Buffers and solutions
Storage solutions . The composition of the buffers and reagents is shown in Appendix 1.
Dilute the storage solution to the appropriate concentration.
0.5XTBE and/or 1XTBE
Gel
Denaturing polyacrylamide gels
Already described in Sections 8, 9, 10
Nucleic acids and oligonucleotides
DNA sequencing reactions
See protocols 3-7 for descriptions.
Specialty Equipment
Automatic Microfiller (20ul)
The microfuge should be equipped with a set of capillary tips (e.g., Multi-Flex) or other spiking equipment (see Sequencing Gel Spiking Step 8).
Bull's Head Clamps (5 cm long, 5-7 per gel)
Gel temperature monitoring strips
These strips are TLC (thermochromic liquid crystal) indicators that change color when the temperature of the gel rises during electrophoresis. Several commercial companies, including BioWhittaker, sell temperature monitoring strips.
Pasteur pipette
Plastic-coated metal plates
Optional. See step 2.
Regulated power supply capable of supplying more than 75W.
Most power supplies with automatic line switching are capable of supplying stabilized voltage, power, or current. A power supply with multiple outputs is capable of delivering 300W of stabilized power at 7000V and can easily perform two or three sequencing electrophoreses simultaneously.
Dissecting Blade with Interchangeable Blades
This blade is used to remove the insulating tape from the sequencing gel mold and sometimes to scrape residual polyacrylamide from the spike plate.
Dissecting blades
The slots on many models of electrophoresis instruments are universal. Although based on the same design, the arrangement of glass plates and gaskets varies between manufacturers. Most commercial equipment runs relatively well, and the choice of manufacturer is largely a personal one. However, the authors recommend that it is best to purchase units with fixed metal plates. These plates, generally made of aluminum, dissipate heat generated by electrical resistance through the gel surface, thus preventing "smiling" artifacts from appearing at the edges of the gel.
Shark tooth combs (0.4 mm thick, with 32, 64 or 96 teeth, depending on the requirements of the electrophoresis apparatus)
It is necessary to use the same tooth comb during the gel molding process.
Syringe (10cc) and 22 gauge hypodermic needle
85°C water bath and 100°C oven
The water bath is used when the sequencing reaction is performed on a microtitre plate. An oven is used when the reaction is performed in a capillary tube.
Methods
Warning: In practice very high voltages are applied to the DNA guillotine gel. Excessive current can cause severe burns, fibrillation of the heart muscle, central respiratory arrest, and asphyxiation due to paralysis of the respiratory muscles. Always make sure that: the gel cassettes used for electrophoresis are insulated, all buffer tanks are covered, and the gels are placed on a dry, stable lab bench. Turn off the power before either adding a sample or taking a gel.
1. Wipe any dried polyacrylamide off the gel mold with a damp paper towel or a damp sponge. Pipette a few milliliters of 1XTBE buffer solution along a smaller or cut glass plate to add it, slowly removing the comb from the gel as you go. Use a dissecting blade to cut the insulating tape and peel it away from the bottom of the gel mold. Do not remove it from the edge of the gel.
2. Secure the gel mold to the electrophoresis apparatus with bullhead clamps according to the manufacturer's instructions, using plastic-coated laboratory clamps or with the screw clamps inherent in the electrophoresis apparatus. Generally smaller or notched plates should be in direct contact with the electrophoresis instrument, while larger ones without slots should be directed toward the observer.
During electrophoresis, if the electrophoresis machine does not have a metal plate to dissipate the heat generated by electrophoresis, a metal plate, such as a plastic-coated aluminum plate, cut to the same size as an ungrooved glass plate will dissipate the heat very well and is lightweight.
3. Fill up the upper and lower tanks of the electrophoresis apparatus with suitable buffer.
For standard or formamide-containing gels
a. Fill the upper and lower tanks with 1XTBE. The height of the buffer in the lower tank should be just above the bottom of the glass plate. The upper tank should be just above the top edge of the plate with the smaller incision and in direct contact with the gel.
b. Rinse the top of the gel with a 10 ml syringe filled with lxTBE to remove excess polyacrylamide and urea from the gel. If necessary, scrape all polyacrylamide debris from the glass plate with the syringe needle.
Use a Pasteur pipette to dislodge any air bubbles attached to the bottom of the lower trough glass plate.
c. Connect the electrodes to the electrophoresis unit and supply power. The cathode (black) should be connected to the upper tank and the anode (red) to the bottom, with the inherent temperature receptors (if any) or temperature monitoring strips attached. Electrophoresis at a constant voltage of 50~70W for about 45 min or until the temperature of the gel reaches 45~50°C. Disconnect the power supply and detach the electrodes.
For electrolyte gradient gels
a. Fill the upper tank with 0.5 XTBE and the lower tank with a mixture of 2x Hovenia 1XTBE plus 1x the volume of sodium acetate (see Scheme 10).
b. Rinse with 0.5XTBE as above to remove urea/polyacrylamide. Do not pre-electrophoreze electrolyte gradient gels.
4. Incubate the capillary containing the sequencing reaction in an oven at 100°C for 2 min. If the reaction has been completed on a microtitre plate open the cap of the microtitre plate and float the uncovered titre plate in a warm bath at 85°C for 5 min.
5. While incubating the titration plate, fill an l0CC syringe with 0.5X or 1XTBE with a 22-gauge needle and forcefully push out a few sprays of TBE onto the gel surface submerged in the electrophoresis solution used for spiking in order to remove any residual urea and polyacrylamide debris from the spiking zone. Continue pushing until no residual urea is visible in the spiking zone.
Rapid diffusion of urea from the gel will form mats at the bottom of the wells and narrow slits. Due to the higher concentration than the sequencing reaction . The urea mat will replace the reactants at the bottom of the wells. The result of this displacement is an uneven migration of DNA fragments into the gel and an irregular sequence ladder.
6. Gently insert the shark-tooth comb into the spiking well (teeth down) and push the shark-tooth comb down until the teeth just penetrate the surface of the gel.
7. Remove the filament tube or microtitre plate cover from the water bath or oven to ice. Keep the sample at 0°C until it is spiked onto the gel. rapid chilling to low temperatures is to delay the restitution between the template and the radiolabeled strand.
8. 1 to 5 ul of each sequencing reactant (see volumes described in Schemes 3 to 7) is spiked into an adjacent slot on the gel.
See Sequencing Gel Spiking.
The sample will flow into the wells to form a tight band on the polyacrylamide surface. Record the order of the templates and take care that each set of samples is spiked in the same order.
An old copy of the sequencing reaction can be added to the extra spiking slot at the edge of the gel as a boundary in order to accurately and unambiguously separate the left and right orientations of the final radioautographic film. 
9. When all samples have been added, connect the power supply to the electrode device: negative (black) to the upper tank and anode (red) to the lower tank. Gel electrophoresis should be performed at a constant enough power to maintain a temperature of 45 to 50°C. The gel electrophoresis should be performed at a constant enough power to maintain a temperature of 45 to 50°C. 
10. Depending on the distance between the target sequence and the oligonucleotide primers, a second addition is made when the bromophenol blue in the buffer of the first addition migrates out of the standard or formamide-containing denaturing polyacrylamide gel after about 15 min (1.5-2.0 h), and the sequencing reactants are again added to the gel. The sequences from the first spiking are farther away from the primers, while the sequences from the second spiking are closer. This second spiking method extends the length of the readable sequence by about 35%.
a. Turn off the power and separate the sequencing unit from the power supply.
b. Remove the electrophoresis buffer from the upper and lower tanks.
c. Heat denature the samples as described in step 4 above.
d. Add sample.
e. Reconnect the sequencing device to the power supply.
f. Perform electrophoresis as before, maintaining the temperature at 45-50°C at a sufficiently constant power.
As an alternative, the length of the readable sequences can be extended by replacing the electrophoresis buffer in the lower bath with a buffer consisting of 2 parts 1XTBE and 1 part 3mol/L sodium acetate at an appropriate stage of the electrophoresis process.
11. At the end of the electrophoresis, the gel is removed and radiographically autoradiographed according to the procedure described in Scheme 12.