Molecular Techniques and Methods

Preparation of Sequencing Gels

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION

DNA sequencing involves a specific application of electrophoresis to resolve the linear single-stranded fragments produced during sequencing reactions, which differ in length by a single base pair. This necessitates using an acrylamide gel, usually at a concentration of 4-20%, of at least 40 cm in length and normally 0.4 mm thick.

  • Sequencing gel solutions are poured into a mold comprising two glass plates held apart by plastic spacers that ran the length of the plates at their edges.

  • The wells into which samples are loaded may be formed by the "shark's tooth" comb, which has a straight edge and a jagged edge comprising 24-48 triangular teeth.

  • The radiolabeled fragments produced during the four chain-termination reactions are run on adjacent lanes of the gel. Several steps must be taken to prevent DNA from forming stable secondary structures by self-hybridization. The samples are heated to at least 70oC in the presence of the denaturant formamide before loading, the denaturant urea is incorporated into the acrylamide gel at 7M, and the gel is run at around 50oC.

  • The samples undergo electrophoresis for an appropriate length of time, which is determined by the distance between the primer used to prime the sequencing reactions and the region of sequence to be analyzed. The gel is then dried and exposed to X-ray film. A sequence "ladder" is produced from which the sequence of the DNA template can be determined by reading successive bands of increasing size in the four adjacent tracks of the gel.




    MATERIALS AND SOLUTIONS

    30% Acrylamide:Bis Solution (29:1) (100 ml)
    Acrylamide ------------------------------------------ 28 g
    bis-Acrylamide -------------------------------------- 1 g
    Add deionized H2O to make a final volume of ----- 100 ml
  • Filter through 0.4 um filter.
  • Store at 4oC.


    Gel Fixative (100 ml)
    10 % Glacial Acetic Acid ---------------------------- 10 ml
    10 % Methanol -------------------------------------- 10 ml
    Distilled H2O ---------------------------------------- 80 ml




    PROCEDURES

    Preparation of Sequencing Gel

    1. Clean glass plates thoroughly with 0.01% SDS, distilled H2O, Glass Cleaner, and then ethanol.

    2. Siliconize one glass plate by siliconizing solution (dimethyl dichlorosilane).
  • This will help to remove the gel from the glass plate after electrophoresis.
  • Do not siliconize both glass plates.

    3. Assemble the glass plates and spacers.
  • Glass plates: 21-38 cm wide x 40-100 cm long
  • Spacers: 0.4 mm thick

    4. Combine the following solutions in a beaker.

    4%
    6%
    8%
    Urea
    63 g
    63 g
    63 g
    5 x TBE
    30 ml
    30 ml
    30 ml
    30% Acrylamide:Bis Solution (29:1)
    20 ml
    30 ml
    40 ml
    Add distilled H2O to make a final volume of
    150 ml
    150 ml
    150 ml

  • This solution can be made up as a stock and stored at 4oC for several weeks.


    5. Mix the following solution and pour between the plates immediately.

    Urea/TBE/Acrylamide Gel Mix from step 4
    50 ml
    10% Ammonium persulfate
    125 ul
    TEMED
    50 ul


    6. Insert the straight edge of the shark's tooth comb about 5 mm into the gel.
  • It is advisable to clamp the glass plates over the comb with two bulldog clips to reduce the risk of leakage across adjacent lanes during sample loading.

    7. The gel should set within 1 hour, but to maximize resolution, it is recommended to age the gel for at least 3 hours before use.
  • If the gel is to be left overnight, place a moistened paper towel over the comb, and cover the upper end of the assembly with cling film to prevent the gel from drying out.



    Running the Sequencing Gel

    8. Assemble the gel apparatus and pour 1 x TBE (pH 8.3).

    9. Load 5 ul of the Formamide Indicator Dye, which is used to stop the sequencing reactions, into a few of the wells, and pre-run the gel at an appropriate voltage (e.g., for an 21 x 50 cm 8% gel; 2,000 V/ 50 W) for 20-60 min until the temperature stabilizes at 55oC.
  • It is important not to let the temperature exceed 65oC, since this may hydrolyze the gel or cause the glass plates to crack.

    10. Denature the sequencing reaction samples into single strands by heating to 95oC for 3 min.
  • Immediately plunge them into ice to prevent reannealing.

    11. Turn off the power supply, and rinse the loading wells with 1 x TBE.
  • Load 2-3 ul of each of the four termination reactions from each template into adjacent wells of the gel.

    12. Run the gel at 50oC until the sample dyes have migrated the required distance.

  • Bromophenol blue - migrates with a DNA fragment of approx 26 nucleotides.
  • Xylene cyanol - migrates with a DNA fragment of approx 106 nucleotides.


    Gel Dryng and Autoradiography

    13. At the end of the run, disconnect the power supply and remove the gel.
  • Rinse buffer off the gel, and discard buffer from the gel apparatus into a sink designated for liquid radioactive waste.
  • Remove any clamps or clips that secured the plates together during the run.

    14. Remove the siliconized plate by gently prising the plates apart at one end.
  • The gel should adhere to the other plate.

    15. Optional: Fix the gel in the Gel Fixative for 15 min on one glass plate.
  • This procedure removes urea and decreases the time required for drying.

    16. Cut a piece of Whatmann 3MM paper to the appropriate size, and gently lay it on top of the gel.
  • Rub the back of the Whatmann paper with a paper towel, and then peel it away from the glass plate. The gel will remain stuck to the Whatmann paper.

    17. Cover the gel with cling film, smoothing out air bubbles with a paper towel.

    18. Dry the gel in a stab gel dryer at 80oC for 2 hours until the gel is dry.

    19. Remove the cling film (this is essential with 35S because it is such a weak emitter, but not necessary with 32P), and autoradiograph the gel against a X-ray film in a cassette.




    NOTES

  • Unpolymerized acrylamide is a neurotoxin. Gloves and mask should be worn, and care should be taken when handling acrylamide powder or solutions.

  • Leaks and air bubbles constitute the most problematic aspect of successful gel pouring. The key to avoiding these is thorough cleaning of gel plates and spacers.

  • It is advisable to distinguish the two sides of each glass plate to ensure that the same side is always used for the inner ("gel") and outer sides, and that it is always the same side of one of the plates that is siliconized.

  • The concentration of acrylamide to be used depends on the distance of the sequence to be resolved from the sequencing primer. A 6% gel is suitable for reading between 25 and 400 nucleotides from the primer. Higher concentrations (12-20%) may be used for sequences within 50 nucleotides, and lower concentrations (4 or 5%) for >400 nucleotides.


    KIT INFORMATION



    REFERENCES

  • Biggin, MD, Gibson, TJ, Hong, GF (1983) Buffer gradient gels and 35S label as an aid to rapid DNA sequence determination. PNAS 80: 3963-3965.

  • Reed, AP, Kost, TA, Miller, TJ (1986) Simple improvements in 35S dideoxy sequencing. BioTechniques 4: 306.

  • Wahis, WP, Kingzette, M (1988) No runs, no drips, no errors: a new technique for sealing polyacrylamide gel electrophoresis apparatus. BioTechniques 6: 308.

  • Please send your comment on this protocol to "editor@MolecularInfo.com".

  • Home
    MT&M
    Online Journal
    Hot Articles
    Order Products
    Classified