92
KAI-OLAF NETZER
visable to first pour a gel with a wide gradient range (0-80%). Any other
concentration range may be achieved by adjusting the composition of
the two stock solutions (see step 7B. below).
9A. Add 70 JlllOo/o ammonium persulfate to each tube, mix gently, and
add 7 Jll TEMED. Mix gently.
lOA. Carefully pour the high er concentration stock solution into the chamber of the gradient maker which is next to the exit. Open the stopcock
connecting the two chambers so that the solution fills the closest passageway, then close again. Add the lower concentration stock solution
to the second chamber. Place the gradient maker on a magnetic stirrer
elevated 25 cm above the bench. Stir the chamber next to the exit at
medium velocity. Mixing should not introduce bubbles into the solution. Insert the 20 gauge needle in the 5 mm opening at the top end of
the gel. Carefully open the stopcock in between the two chambers and
the exit of the gradient maker. Adjust the flow so that the gel is poured
within 4 minutes.
llA. Allow one hour for polymerization at room temperature.
12A. Preheat approximately 5 1 of lx TAE buffer (for Hoefer Scientific
SE600) to 60°C in a microwave oven.
Note: The buffer can be reused 3 tim es.
13A. Preheat the water bath to approximately 70°C. Connect the external
circulation with the heat exchanger of the electrophoresis unit as soon
as the tank is filled with buffer.
14A. Place the buffer tank on the magnetic stirrer. Stir at medium velocity.
Insert a thermometer into the tank and adjust the buffer temperature
to 60 oc.
ISA. Remove the third spacer from the gel. If necessary, apply silicone
grease to form a complete well at the top of the gel. Disassemble
the casting apparatus and insert the gel into the tank. Flush the
weil with running buffer using a syringe and 20 g needle.
Note: Wait 10 min for temperature equilibration before loading the gel.
16A. Dilute approx. lJlg DNA in 500 Jlllx TAE. Add 100 J.1l6x gelloading
buffer. Load the sample into the gel.
17 A. Run the gel for approximately 500 Vh. Recirculate the buffer using a
peristaltic pump (10 mVmin).
KAI-OLAF NETZER
visable to first pour a gel with a wide gradient range (0-80%). Any other
concentration range may be achieved by adjusting the composition of
the two stock solutions (see step 7B. below).
9A. Add 70 JlllOo/o ammonium persulfate to each tube, mix gently, and
add 7 Jll TEMED. Mix gently.
lOA. Carefully pour the high er concentration stock solution into the chamber of the gradient maker which is next to the exit. Open the stopcock
connecting the two chambers so that the solution fills the closest passageway, then close again. Add the lower concentration stock solution
to the second chamber. Place the gradient maker on a magnetic stirrer
elevated 25 cm above the bench. Stir the chamber next to the exit at
medium velocity. Mixing should not introduce bubbles into the solution. Insert the 20 gauge needle in the 5 mm opening at the top end of
the gel. Carefully open the stopcock in between the two chambers and
the exit of the gradient maker. Adjust the flow so that the gel is poured
within 4 minutes.
llA. Allow one hour for polymerization at room temperature.
12A. Preheat approximately 5 1 of lx TAE buffer (for Hoefer Scientific
SE600) to 60°C in a microwave oven.
Note: The buffer can be reused 3 tim es.
13A. Preheat the water bath to approximately 70°C. Connect the external
circulation with the heat exchanger of the electrophoresis unit as soon
as the tank is filled with buffer.
14A. Place the buffer tank on the magnetic stirrer. Stir at medium velocity.
Insert a thermometer into the tank and adjust the buffer temperature
to 60 oc.
ISA. Remove the third spacer from the gel. If necessary, apply silicone
grease to form a complete well at the top of the gel. Disassemble
the casting apparatus and insert the gel into the tank. Flush the
weil with running buffer using a syringe and 20 g needle.
Note: Wait 10 min for temperature equilibration before loading the gel.
16A. Dilute approx. lJlg DNA in 500 Jlllx TAE. Add 100 J.1l6x gelloading
buffer. Load the sample into the gel.
17 A. Run the gel for approximately 500 Vh. Recirculate the buffer using a
peristaltic pump (10 mVmin).