Experiments on the preparation of denaturing polyacrylamide gels
Experiments on the preparation of denaturing polyacrylamide gels
In sequencing, 4 series of DNA fragments are analyzed by electrophoresis on a thin polyacrylamide gel under denaturing conditions. This protocol describes the preparation of a homogeneous buffer and acrylamide concentration gel. This experiment was derived from the next volume of the Laboratory Guide to Molecular Cloning (Third Edition) by [American] J. Sambrook D.W. Russell.
Operation method
Experiments on the preparation of denaturing polyacrylamide gels
Materials and Instruments
Acrylamide solution Aqueous ammonium persulfate solution Deionized water Decontaminants Ethanol KOH Methanol solution Polysiloxane solution TBE Electrophoresis buffer TEMED Urea Move makings For more product details, please visit Aladdin Scientific website.
Spring clips Dry glue holders Sealing tape Glue boards (paired) and dividers Gloves Vaseline Protective work compound Shark tooth combs Flasks with arms Spacers Syringes Test tube holders
Buffers and solutions
Prepare storage solutions, buffers, and reagents by referring to Appendix 1. Dilute the storage solution to the appropriate concentration.
Acrylamide solution (45% m/V)
Acrylamide (DNA sequencing grade) 434 g
N,N'-methylenebisacrylamide 16 g
Add water to 600 ml
Heat to 37°C to promote dissolution Make up to 1 liter with distilled water and filter through a nitrocellulose membrane (pore size 0.45 ptn). Store in a brown bottle at room temperature. A more expensive alternative is to purchase pre-mixed acrylamide/bisacrylamide powder and dissolve it in water. Cheaper acrylamide/bisacrylamide powders are usually mixed with metal ions. The resulting storage solution needs to be purified by mixing it with 0.2 volume of resin (MB-1, Mallinckrodt), stirring, and filtering through Whatmenl paper. During storage, acrylamide/bisacrylamide slowly becomes acidic, a denitrification process catalyzed by light and alkali. Keep the solution at pH 7.0 or below and store in a black bottle at room temperature. It should be reconstituted every few weeks.
Ammonium Persulfate Aqueous Solution (1.6% m/V)
Deionized water
Decontaminant
Ethanol
KOH/methanol solution
Prepared by adding 5 g of K0 H to 100 ml of methanol, used for cleaning glass plates and stored in airtight glass vials.
Polysiloxane solution
Traditional polysiloxane solutions include dichlorodimethylsilane, which is toxic, volatile and flammable. In recent years, a number of non-toxic alternatives have emerged, including Gel Slick (FMC Bioproducts), RainX (Unelko, Scottsdale Arizona), and Acrylease (Stratagene).
10XTBE Electrophoresis Buffer
TBE is used in polyacrylamide gel electrophoresis at a concentration of lx (89 mmol/L Tris-boronic acid, 2 mmol/L EDTA), which is twice the amount used for agarose electrophoresis (see Chapter 5). Vertical electrophoresis tanks for polyacrylamide gel electrophoresis generally have a small buffer pool, so the amount of current passed is usually quite large. It is necessary to use lxTBE to ensure a proper buffer capacity. The pH of the buffer should be close to 8.3. pH adjustments are not normally necessary; however, the pH should be carefully checked with each new 10XTBE electrophoresis buffer.
Use the same 10xTBE electrophoresis buffer for both the gel and the working buffer, as small differences between the two can cause severe distortions in DNA migration.
The use of sulfamic acid (36 g/L 10XTTE buffer) in place of boric acid in the standard TBE solution reduces the distortion of bands at the top of the gel due to the formation of glycerol-boronate anionic lipids (Pisa-Willanson and Fuller 1992). See the section on Glycerol in DNA Sequencing Reactions if more information is needed.
TEMED (N,N,N',N'-tetramethylethylenediamine)
Electrophoresis grade TEMED is available from many companies (Sigma, Bio-Rad). TEMED is hygroscopic and must be stored at 4°C in an airtight bottle. It is a linking catalyst in polymerization reactions.
Urea, solid
Specialty devices
Spring clips: 5 cm long, 5 to 7 per piece of rubber.
Glue drying rack
Although not important, a dry glue rack is very convenient for drying and storing glass plates for sequencing (Bio Whitraker).
Sealing Tape
Examples include 3M Scotch Stretchable Tape (Lab Safety Supply, Janesville, Wisconsin), 3M Scotch yellow electrical tape #56 (Life Technologies), 3M Scotch polytetrafluorethylene (PFTE) extruded film tape. see Hengen (1996) for a discussion of the use of different tapes and other sealing methods.
Adhesive sheets (paired) and spacers
The gluing boards are either slightly longer than the other board by 3.5-4.0 cm, or one board is not engraved. To avoid cracked or leaking plates, it is best to keep the plates paired and corresponding to the slots of the sequencing gel.
Gloves
Talc-free, easy-to-handle rubber or polyvinyl chloride (PVC).
Vaseline
Optional, see step 4
Protective working hinge paper
Plastic on one side (Kaydry Lab Cover from Fisher) or Benchkote.
Shark tooth comb
0.4 mm thick, 32, 64 or 96 teeth. Dependent on the capacity of the electrophoresis unit.
Flasks with arms (250 ml)
Separators (uniform thickness or narrow edges)
Two plates per glue, made of thin flexible plastic sheets (0.4 mm) or Teflon (Sanger and Coulson 1978), separate the glass plates. An impermeable seal is formed between the adhesive plates and the spacers so that the unconsolidated adhesive solution cannot escape.
The narrow edges of the spacer are used to create a glue that is thicker at the bottom than at the top, resulting in a narrower band at the bottom and a more uniform band throughout the glue. Despite this advantage, it is more difficult to prepare and dry.
Syringe (60cc)
Optional, see step 15.
Test Tube Rack
55°C water bath
Method
Important: To prevent contamination with skin oils. Talc-free gloves must be worn at all times and only the edges of the plate should be held.
Preparation of the adhesive plate
1. If necessary, shake KOH/methanol to clean old stains from the plate.
2. Then wash the glass plate and spacer with a warm stain remover solution, rinse thoroughly with tap water, and rinse with deionized water. Rinse the glass plate with ethanol to remove watermarks and set aside to dry.
The glass plate must be washed carefully. This is to ensure that no air bubbles are created during gluing.
3. Treat the inside of the small glass panes with a polysiloxane solution. In a chemical fume hood, place the glass plate on a stack of paper towels, inside surface up, and pour a small amount of silanizing solution over it. Several sheets of Kimwipes were used to spread the silanizing solution over the surface of the glass plate and the plate was allowed to air dry (l to 2 min). The glass plate was then washed with deionized water, then with ethanol and air dried.
4. Place the large glass plate (clean side up) on an empty test tube rack and place the spacers on either side of the glass plate (see Figure 12-9).
If using spacers with narrow edges, place the thicker side on the bottom of the plate. Add a small drop of petroleum jelly between the large plate and the spacer so that the spacer does not move during the next step.
5. Place the smaller (or notched) glass plate over the spacer again, aligning the spacer properly.
6. Clamp one side of the glass plate with several large spring clips (5 cm long).
Apply gel sealing tape to the other side of the glass pane and to the bottom of the glass pane to form an impermeable seal. Special attention must be paid to the bottom corners of the glass pane as these are the most vulnerable to leakage.
7. Replace the spring clamps to the sealed side and apply gel seal tape to the other side of the glass panel.
8. Place a comb on the open end of the mold and check that the apricot fits, remove the comb and place the empty mold on the lab table. 
Gel Dispensing
9. Cover the work area of the table with plastic-lined protective paper.
It is almost impossible to avoid dripping acrylamide solution on the table when filling sequencing gels.
10. Prepare the appropriate concentration of acrylamide solution (Table 12-19) in a 250 ml arm conical flask. This is enough solution to prepare a 40 cm x 40 cm sequencing gel.
Note: The gel must be prepared in a single step with no interruptions.
11. Mix all reagents and heat in a 55°C water bath for 3 min to help dissolve the urea.
The process of dissolving urea is very slow and must rely on external heat. The approximate volume is 66 ml, add water to 100 ml.
12. Remove the solution from the water bath and cool for 15 min to room temperature. Stir the mixture continuously.
13. Place the flask in a vacuum device to evacuate the gas.
This prevents gas bubbles from forming during polymerization.
14. Place the solution in a 250 ml glass beaker. Add 3.3 ml of fresh 16% ammonium persulfate and mix well.
Older ammonium sulphate does not polymerize well enough and will produce faint bands. 
15. Add 50ulTEMED and gently swirl the container to mix the solution. Dispense the gel directly. Alternatively, use a 60cc syringe to draw up about 40 ml of the above solution. be careful that there are no air bubbles.
Compared to protein electrophoresis, the largest amount of TEMED has been used to ensure that the polymerization is fast enough and homogeneous enough. The speed of polymerization is time dependent, the lower the temperature the slower the polymerization. Experienced people can make multiple 40 cmx40 cm gels from a single preparation by pre-cooling.
From this point on, move as quickly as possible.
16. Hold the mold with your hand at an angle of approximately 45° to the horizontal and slowly pour the solution from the pipette along one side (see Figure 12-9). 
17. Place the mold on the test tube rack (see Figure 12-9).
This position reduces the water pressure at the bottom of the mold and prevents glue leakage.
18. Immediately insert the flat side of a shark-tooth comb approximately 0.5 cm into the gel solution, with both ends of the comb inserted at the same depth into the liquid so that the flat surface of the comb remains level when the gel is placed vertically.
If bubbles are visible next to the comb, slowly remove the comb. Wash the surface of the comb and reinsert it into the gel.
19. Hold the comb in place with the spring clamp. Add the remaining acrylamide-urea solution along the top of the gel to form a string of acrylamide droplets. Allow the gel to polymerize for 15 min at room temperature.
20. Flush the 60cc syringe so that it does not become clogged with polymerized acrylamide.
WARNING: A small amount of unpolymerized acrylamide will be released during flushing; gloves should be worn.
21. Inspect the gel after 15 minutes of polymerization for a Schlieren line of varying refractive index just below the flat surface of the comb, which is an indication of satisfactory polymerization. When polymerization is complete (approximately 1 hour), remove the spring clip.
WARNING: A small amount of unpolymerized acrylamide will be released during rinsing, gloves should be worn.
22. After polymerization, the gel can be used immediately (see Option 11) or stored at room temperature for up to 24 hours or at 4°C for 48 hours. To prevent dehydration during storage, leave the comb inside the gel and place some 1XTBE-moistened paper towels around the top of the gel, which can be covered with Saran wrap. Do not remove the comb at this stage. 