DNA?Sequencing

Pouring the Gel

Outline:

Pouring this big & thin 6% acrylamide gel ("Mother of all gels") is quite a challenge and probably the reason why smart whimps by them ready to use.

Supplies & Equipment:

waterbath 37℃

Erlenmeyer flask 250 mL with rubber stopper and vaccum adapter

vacuum source

funnel & Whatman filter paper

BIORAD sequencing gel apparatus

beakers (50 mL and 100 mL)

syringe (60 mL) with large needle

styrofoam box (inclined)

styrofoam box (rectangular)

gel clamps (6)

food wrap

Reagents:

AquaSil (SigmaCoat)

Temed

3 MM paper (bottom tray size)

Procedure:

Weigh urea in 250 mL Erlenmeyer flask. Add acrylamide, TBE and water (total volume 135 mL). Place in 37℃ waterbath to dissolve urea (do not overheat!).

Clean the glass plates, spacers, comb of the gel apparatus - first with water, then with 70% Ethanol. Siliconize bottom plate by wiping with AquaSil. Let air dry for 5 minutes. Assemble Gel apparatus (plates should be perfectly aligned at the bottom, spacers should stick out just 1 mm). Place two pieses of 3 MM paper in the bottom of the seal casting tray. Keep this tray in a cool place (4℃) until used.

When urea is dissolved, add 75 mg ammonium persulfate, mix to dissolve.

Connect flask to vacuum and degas for 20 minutes (rewarm solution if crystals reappear).

Filter through Whatman filter paper in a refrigerator (4℃).

Transfer 30 mL into small beaker, add 100 µL icecold TEMED, mix quickly and pour onto the 3MM paper strips in the (chilled) bottom tray. Quickly place gel apparatus in tray to seal bottom. Let polymerize in a fixed position for 20 minutes.

When seal is polymerized, place on an inclined styrofoam box. Insert a paper towel in the apparatus, preventing excessive acrylamide solution from spilling in the buffer space.

Add 40 µL icecold TEMED to the remaining (chilled) acrylamide solution, mix, aspirate in 60 cc syringe and pour gel immediately. Insert the back of the shark comb about 7 mm into the gel to form a straight edge. Clamp the two glass plates onto to comb, moisten the paper towel or additional kimwipes with water. Cover the top of the gel apparatus with food wrap keep moist (especially when polymerizing overnight). Place gel leveled on rectangular foam box. Let polymerize for 2 hours (best overnight).

When ready for electrophoresis, remove the shark tooth comb and rinse the gel surface with 1 x TBE. Clean and reinsert the comb with the teeth barely touching the gel surface. Set up the gel on the buffer tank. Fill with 1 x TBE, and pre-electrophorese for 30 minutes at 2500 V (until the gel is warm). Label the lanes that will be used on the glass plate.

Time Required: 4 hours

Sequencing Reaction

Outline:

Sequencing by the chain-termination method involves the synthesis of a DNA strand by a DNA polymerase using a single stranded template. Synthesis is initiated at the site where an oligonucleotide primer anneals to the template. The synthesis reaction is terminated by the incorporation of a nucleotide analog (ddNTP) that terminates elongation. When proper mixtures of dNTP's and one of the four ddNTP's are used, polymerization will be terminated randomly at each possible site. Alternative: Thermo Sequenase radiolabelled terminator cycle sequencing kit (Amersham Life Science #79750)

Supplies & Equipment:

wet ice

waterbath at 68℃

microfuge

heat block or waterbath at 37℃

heat block or waterbath at 90-100℃

Reagents:

Sequencing Kit (Sequenase Version 2.0, Amersham Life Science #70770)

Sequenase 2.0 T7 Polymerase

Sequenase dilution buffer

Termination mixes for dGTP sequencing (ddGTP, ddATP, ddTTP, ddCTP)

dGTP labeling mix

TDMN Buffer (0.28 M TES (free acid), 0.12 M HCl, 0.05 M DTT, 0.08 M MgCl2 , 0.2 M NaCl), TES = N-tris [hydroxymethyl]methyl-2-aminoethane sulfonic acid, MW 229.2

Stop solution

1 N NaOH (freshly prepared from 10 N Stock)

[35 S]dATP (1000 Ci / mmol)

Procedure:

Denature DNA sample for 10 minutes at 68℃.

Neutralize the DNA solution by adding 4 µL of TDMN solution . Allow to anneal by slowly cooling down to room temperature over 30 minutes. Longer annealing time is beneficial for some primers.

While the annealing mixes are incubating, prepare 4 tubes per DNA sample, each containing 2.5 µL of one of the four termination mixes (ddNTP's: G, A, T, C). Prewarm at 37℃ in an incubator.

To each annealed DNA sample (Step #2), add the following:

3.0 µL 1 x Labeling mix (dilute 5 x stock with water)

1.0 µL [35 S]dATP (1000 Ci / mmol).

Allow to stand at room temperature until used.

Dilute the sequenase 1:8 (= 1.5 units / µL) with Enzyme Dilution Buffer and store on ice. For n DNA samples, prepare 2 (n + 1) µl of the dilution.

Transfer two sets of the Termination Mix tubes (Step #3) to a heating block (37℃) with the lids opened.

Start the sequencing reaction of two DNA samples at a time. To two annealed DNA -Labeling Mix (Step #4) at room temperature add 2 µl of diluted Sequenase to start the reaction. (place open tubes in microfuge, pipet sequenase to the top of each tube, start the reactions all at once by spinning for 1 second, remove from microfuge.) Incubate at room temperature for 2 minutes. Mix gently during the incubation period.

When the 2 minute-incubation is up, transfer 4.0 µL of each sequencing reaction mix (Step #7) to each of the four (G, A, T, C) pre-warmed (37℃) Termination Mix tubes (Step #6) in the same group. Be sure to change the pipet tip each time. Close the caps, mix briefly (no need to spin) and return immediately to the heating block. Continue to incubate at 37℃ for 10 minutes. Note: Do not start the termination reaction at temperature below 37℃, or the enzyme will pause giving a band in all four lanes.

Transfer the tubes to ice bucket. Add 5 µL of sequenase stop solution, mix then spin for 1 second.

Repeat Steps 6 - 9 until all samples are done.

Heat heat all tubes at 90℃ for 3 minutes. Quickly chill in ice. Store at -20℃ until use.

Time Required: 40 minutes

Electrophoresis

Outline:

Sequencing electrophoresis is performed at high voltage (800 - 2400 V). 1000 V is optimal for high resolution but takes longer. Two dyes, contained in the termination mix give orientation. The first (lower) band is bromophenol blue, the second (upper) band is green (xylene cyanol).

Supplies & Equipment:

power supply

BIORAD sequencing apparatus

gel dryer (hooked to vacuum)

large x-ray film, cassette, film developer

freezer -80℃

Reagents:

1 x TBE (diluted from 5 x stock)

Whatman 3 MM (gel size)

food wrap

Procedure:

When ready for electrophoresis, remove the shark tooth comb and rinse the gel surface with 1 x TBE. Clean and reinsert the comb with the teeth barely touching the gel surface. Set up the gel on the buffer tank. Fill with 1 x TBE, and pre-electrophorese for 30 minutes at 2500 V (until the gel is warm). Label the lanes that will be used on the glass plate.

Turn off the power, push the comb a little deeper, rinse the sample slots with buffer using a 60 mL syringe, load 1.5 to 2 µL sample per slot on the sequencing gel (G, A, T, C). Do not load more than 2 reactions (8 lanes) at a time and 'lay' the sample as close to the gel surface as possible, to minimize diffusion. Run the sample into the gel at 1000 V until dyes separate.

Let first loading run until green (upper, second) dye disappears in the bottom of the gel. Ideally, this is performed at 800 to 1000 V overnight. 2400 V is possible, but results in lower resolution.

Let the second loading run until the blue (lower, first) dye disappears in the bottom of the gel. Usually, this is performed at 1000 - 1800 V the next morning. 2400 V is possible, but results in lower resolution.

Carefully separate the two glass plates (siliconized bottom plate on top). Remove comb and spacers. Transfer gel to a piece of Whatman 3 MM paper by placing the paper on the gel and improving contact to the gel by gently rolling with pipet. Carefully lift the paper with the gel attached while (optional) squirting a little bit of water between the gel and glass plate. Cover the gel surface with food wrap.

Place in vacuum gel dryer with paper side down and dry at 80℃ for two hours.

Remove food wrap, expose the gel to x-ray at -80℃ for 2 to five days.

Time Required: setup in evening, run first loading overnight, second loading and gel processing all next day.