Polyacrylamide Gel Electrophoresis (PAGE) of Cycle Sequencing Products

1. Carefully clean a set of gel plates with a detergent that does not leave a residue, wash with distilled water and wipe clean with ethanol.

2. Coat one side of the small plate with repelcote. If possible, mark the plate the first time it is used, with a diamond-tipped pen, and always repelcote the same side.

3. Put the plates together with the repelcoted side on the inside and the spacers at either side.

4. Check that the sharkstooth combs fit tightly between the plates and then tape the plates on either side and at the bottom.

5. Mix gel solution: (NOTE. ACRYLAMIDE IS TOXIC. ALWAYS WEAR GLOVES)

125 ml 0.5x TBE gel mix
250 µl TEMED
250 µl 25% ammonium persulphate (AMPS)

Note: the gel will start setting within approximately 5 minutes of the addition of the AMPS.

6. Pour the gel solution between the glass plates using a 50 ml syringe.

7. Insert combs upside down (i.e., flat side next to gel and nearly as far in as the holes in the comb), and clamp plates tightly using clips.

8. Leave to set for at least one hour. The gel can be stored at 4°C overnight if the top is kept moist with 1 x TBE and covered with cling film.

9. Pre-running the gel:

• remove the combs carefully - the teeth break easily.
• remove the tape.
• rinse the top of the gel with distilled water or 1 x TBE.
• Insert combs with sharks tooths forming wells (with points just touching top of gel).
• Put gel/plate assembly into the gel tank and clamp tightly.

10. Prepare 1000 ml 1 x TBE and pour into top and bottom tanks.

11. Pre-run the gel at a constant 70 watts for one hour.

12. Heat the samples at 95-100°C for two minutes just prior to loading. Wash urea (leaches out of gel during polymerisation) out of wells with a 50 ml syringe and bent needle immediately prior to loading samples.

13. Add 3 µl of each sample to the wells formed by the combs following the order T, C, G, A.

14. Run at a constant 70 watts for approximately two hours (the bromophenol blue marker should just run off the end of the gel into the buffer).

15. Remove the plates from the tank, carefully remove the combs and split the glass plates apart. The gel should remain attached to the larger glass plate.

16. The gel may be fixed in at tray of 10% acetic acid, 10% ethanol, 80% water for 20-30 minutes but this is not necessary.

17. Remove from fixative and lay a piece of 3MM filter paper over the gel. Carefully peel off with the gel sticking to the 3M paper.

18. Dry down for 45 minutes to 1.5 hours on a slab gel dryer set at 80-85ºC.

19. Mark one corner of the dried gel (e.g., by cutting off a corner). Expose gel to X-ray film in a light tight cassette at room temperature (cut off the corresponding corner of the X-ray film). If using an intensifying screen store at -70°C. Exposure times usually range from 18-48 hours with an intensifying screen and 18-60 hours without, depending on the activity of the radioisotope.

20. Develop autoradiograph and immediately label clearly - particularly with the order of loading (i.e., TCGA).