Radiolabeled sequencing gel preparation, loading, and electr

Thursday, November 20, 2003

Description
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.

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

4% 5% 6%
urea 48 g 48 g 48 g
40% A & B 10 ml 12.5 ml 15 ml
10X MTBE 10 ml 10 ml 10 ml
ddH2O 42 ml 39.5 ml 37 ml
15% APS 500 ul 500 ul 500 ul
TEMED 50 ul 50 ul 50 ul

Urea (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):

termination electrophoresis
reaction polyacrylamide gel conditions
short 5%, 0.15 mm X 50 cm X 20 cm 2.25 hours at 22 mA
long 4%, 0.15 mm X 70 cm X 20 cm 8-9 hours at 15 mA
long 4%, 0.15 mm X 70 cm X 20 cm 20-24 hours at15 mA

10. After electrophoresis, remove the buffer wells, the tape, and pry the gel plates apart. The gel should adhere to back plate. Blot the gel to a 40 cm X 20 cm sheet of 3MM Whatman paper, cover with plastic wrap, and dry on a Hoefer gel dryer for 25 minutes at 80degC
11. Place the dried gel in a cassette and expose to Kodak XRP-1 film.

12. Develop the film for 1-5 minutes in Kodak GBX developer, rinse in distilled water for 30 seconds, fix in Kodak GBX fixer for 5 minutes, and then rinse again in distilled water for 30 seconds. Allow the film to air dry.

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Radiolabeled sequencing gel preparation, loading, and electr