Radiolabeled sequencing gel preparation, loading, and electrophoresis

To prepare polyacrylamide gels for DNA sequencing, the appropriate amount of urea is dissolved by heating in water and electrophoresis buffer, the respective amount of deionized acrylamide-bisacrylamide solution is added, and ammonium persulfate and TEMED are added to initiate polymerization. Immediately after the addition of the polymerizing agents, the gel solution is poured between two glass plates, taped together and separated by thin spacers corresponding to the desired thickness of the gel, taking care to avoid and eliminate air bubbles. Prior to taping, these glass plates are cleaned with Alconox detergent and hot water, are rinsed with double distilled water, and dried with a Kimwipe. Typically, the notched glass plate is treated with a silanizing reagent and then rinsed with double distilled water. After pouring, the gel immediately is laid horizontally and a well forming comb is inserted into the gel and held in place by metal clamps. The polyacrylamide gels are allowed to polymerize for at least 30 minutes prior to use. After polymerization, the comb and the tape at the bottom of the gel are removed. The vertical electrophoresis apparatus is assembled by clamping the top and bottom buffer wells onto the gel, and adding running buffer to the buffer chambers. The wells are cleaned by circulating buffer into the wells with a syringe and, immediately prior to the loading of each sample, the urea in each well is suctioned out with a mouth pipette.

Each base-specific sequencing reaction terminated with the short termination mix is loaded using a mouth pipette onto a 0.15 mm X 50 cm X 20 cm, denaturing 5% polyacrylamide gel and electrophoresed for 2.25 hours at 22 mA. The reactions terminated with the long termination mix typically are divided in half and loaded onto two 0.15 mm X 70 cm X 20 cm denaturing 4% polyacrylamide gels. One gel is electrophoresed at 15 mA for 8-9 hours and the other is electrophoresed for 20-24 hours at 15 mA. After electrophoresis, the glass plates are separated and the gel is blotted to Whatman paper, covered with plastic wrap, dried by heating on a Hoefer vacuum gel drier, and exposed to X-ray film. Depending on the intensity of the signal and whether the radiolabel is 32-P or 35-S, exposure times varied from 4 hours to several days. After exposure, the films are developed by processing in developer and fixer solutions, rinsed with water, and air dried. The autoradiogram then is placed on a light-box and the sequence is manually read and the data typed into a computer.

Protocol

1. Prepare 8 M urea, polyacrylamide gels according to the following recipe (100 ml), depending in the desired percentage:

4% 5%6%urea 48g48g48g40%A&B10ml12.5ml15ml10XMTBE10ml10ml10mlddH2O42ml39.5ml37ml15%APS500ul500ul500ulTEMED50ul50ul50ulUrea (5505UA) is from Gibco/BRL.

2. Combine the urea, MTBE buffer, and water and incubate for 5 minutes at 55deg C and then stir to dissolve the urea.

3. Cool briefly, add the A & B, mix, and degas under vacuum for 5 minutes.

4. While stirring, add the APS and TEMED polymerization agents and then immediately pour in between two taped glass plates with 0.15 mm spacers. (Prior to taping, the notched, front glass plate should be treated with a small amount of silanizing reagent and then rinsed with ddH2O).

5. Insert the well forming comb, clamp, and allow the gel to polymerize for at least 30 minutes.

6. Prior to loading, remove the tape around the bottom of the gel and the well-forming comb. Assemble the vertical electrophoresis apparatus by clamping the upper and lower buffer chambers to the gel plates, and add 1X MTBE electrophoresis buffer to the chambers.

7. Flush the sample wells with a syringe containing running buffer, and immediately prior to loading each sample, flush the well with running buffer using gel loading tips.

9. Load 1-2 ul of sample into each well using a Pipetteman with gel-loading tips, and then electrophorese according the following guidelines (during electrophoresis, cool the gel with a fan):