202
KLAUS DEICHMANN
Filter using a 0.45J.!m disp. fllter and degas with vacuum for 5 min. Ureais
responsible for the denaturing conditions of the gel.
8. Add 60 1.11 TEMED and 600 J.!l ammonium persulfate ( 10%) and swirl to
mix.
9. Pour the solution carefully between the plates, using a 20 ml pipette.
Start on one side and take care to avoid introducing air bubbles; carefully insert the flat side of the shark's tooth combs from the top, about 5
mm deep.
10. Allow the gel to polymerize for at least 2 hours.
11. Mount the plates in the sequencing gel apparatus after removing the
tape; clamp so that the smaller plate faces the upper buffer chamber.
12. Fill upper and lower chambers with 1 x TBE. After this step the TBE in
the upper chamber should be in contact with the top of the gel, and the
TBE in the lower chamber should be in contact with the bottom. The gel
thus forms a conductive bridge between the 2 chambers.
13. Invert shark's tooth combs so that the teeth indentabout 0.5 mm into
the top of the gel.
14. Using a syringe with needle, thoroughly rinse the spaces between the
teeth with 1 x TBE buffer to remove unpolymerized acrylamide and
urea diffusing out of the gel.
15. Pre-run gel at40 W/ca.1500 V forabout 1 hour. Theplates should be hot
after the pre-run.
16. Load half of the first set of denatured sequencing reactions into 4 adjacent wells after washing the wells again with 1 x TBE buffer. Always
start from the left side. Leave 4 empty slots before loading the next 4
samples (see step 18.)
17. Run the gel at 1200-1500 V ( about 30 mA, 43 W). Contin ue electrophoresis
until the xylene cyanol front is justabout to reach the bottom of the gel.
18. Load the second half of the same denatured reactions in the next 4,
adjacent wells and continue electrophoresis as before.
19. Stop electrophoresis when the bromophenol blue of the samples loaded
last reaches the bottom of the gel. This procedure will give you a long
run and a short run, i.e. the first 200 bp are read from the set of samples
loaded last on the gel, the following 200-400 bp are read from the set of
samples loaded first, always reading from the bottom to the top.
KLAUS DEICHMANN
Filter using a 0.45J.!m disp. fllter and degas with vacuum for 5 min. Ureais
responsible for the denaturing conditions of the gel.
8. Add 60 1.11 TEMED and 600 J.!l ammonium persulfate ( 10%) and swirl to
mix.
9. Pour the solution carefully between the plates, using a 20 ml pipette.
Start on one side and take care to avoid introducing air bubbles; carefully insert the flat side of the shark's tooth combs from the top, about 5
mm deep.
10. Allow the gel to polymerize for at least 2 hours.
11. Mount the plates in the sequencing gel apparatus after removing the
tape; clamp so that the smaller plate faces the upper buffer chamber.
12. Fill upper and lower chambers with 1 x TBE. After this step the TBE in
the upper chamber should be in contact with the top of the gel, and the
TBE in the lower chamber should be in contact with the bottom. The gel
thus forms a conductive bridge between the 2 chambers.
13. Invert shark's tooth combs so that the teeth indentabout 0.5 mm into
the top of the gel.
14. Using a syringe with needle, thoroughly rinse the spaces between the
teeth with 1 x TBE buffer to remove unpolymerized acrylamide and
urea diffusing out of the gel.
15. Pre-run gel at40 W/ca.1500 V forabout 1 hour. Theplates should be hot
after the pre-run.
16. Load half of the first set of denatured sequencing reactions into 4 adjacent wells after washing the wells again with 1 x TBE buffer. Always
start from the left side. Leave 4 empty slots before loading the next 4
samples (see step 18.)
17. Run the gel at 1200-1500 V ( about 30 mA, 43 W). Contin ue electrophoresis
until the xylene cyanol front is justabout to reach the bottom of the gel.
18. Load the second half of the same denatured reactions in the next 4,
adjacent wells and continue electrophoresis as before.
19. Stop electrophoresis when the bromophenol blue of the samples loaded
last reaches the bottom of the gel. This procedure will give you a long
run and a short run, i.e. the first 200 bp are read from the set of samples
loaded last on the gel, the following 200-400 bp are read from the set of
samples loaded first, always reading from the bottom to the top.