SSR GEL and Silver Staining Protocol

Paula Marquardt & Craig Echt

Published in:

Echt CS, May-Marquardt P, Hseih M, Zahorchak R. 1996. Characterization of microsatellite markers in eastern white pine. Genome 39:1102-1108 .

comments can be directed to Paula Marquardt at:

USDA Forest Service Research

5985 Highway K

Rhinelander, WI 54501

USA

Phone: 715-362-1121

Fax: 715-362-1166

e-mail: pmarquar@newnorth.net

I. EQUIPMENT:

DNA sequencing unit (35 x 45 cm) & 2000V power supply

Clamps

Lg. plastic trays (4), about 43 x 50 x 8 cm, and one lid

Two rocking platforms

Heat block for microtiter plates. A microplate vortexer is helpful.

II. MOLD ASSEMBLY:

Notes: Bind silane is toxic and should be used in a chemical fume hood. Wear gloves when handling this solution. Use a small piece of vinyl tape on a lower outside corner of the acrylease treated glass gel plate to mark the untreated side and also help distinguish the plates. This helps avoid confusion between plates when using offset plates. The tape can remain in place through several electrophoresis / washing cycles.

1. Wash inner and outer plates well with alconox cleanser. Rinse well with tap water, deionized or distilled water, and ethanol, air dry. Use dedicated sponges for each treatment.

2. Using a kimwipe tissue, coat the inner side of notched or offset plate with acrylease (Stratagene) and allow to dry--I do not treat the top 2 inches of the plate since I feel that the nonstick coating promotes leaking between wells behind the teeth of the comb. Buff well with a kimwipe soaked in ethanol for a clean finish; this takes some elbow grease to get the streaks off of the plate. Change gloves before working with bind silane and take care not to cross contaminate the plates with the two treatments. The acrylease treatment only needs to be repeated every four gels or so.

3. Prepare fresh 1 ml binding solution by making a solution 0.0005-0.001% bind silane (Sigma #M-6514) in 95% ethanol, 0.5% glacial acetic acid. Apply with a kimwipe and coat the inner side of the larger plate with one ml and allow to dry 4-5 minutes. Wipe the plate with ethanol in one direction and then perpendicular to first direction, don't use too much pressure. This treatment needs to be repeated every time.

III. GEL SOLUTION PREPARATION:

Note: Acrylamide is toxic. Wear gloves when handling solution and face mask when weighing out powder. A safer alternative is to buy a premix.

1. Rinse all glassware and plastic ware with d.i. water prior to gel solution preparation and pouring, including the disposable filter unit.

2. Gels are 6% acrylamide, 8M urea, 1X TBE. For each gel, mix together 50 g urea, 15 ml 40% 19:1 acrylamide solution, and 31 ml d.i. water. We use a 4.5% gel for fingerprinting reactions.

Note: (We store aliquots of premixed 40% Acrylogel solution, Gallard-Schleisinger Ind., at -20℃.)

3. Warm and stir the mixture in a beaker of warm water until all the urea is dissolved. Add 1.25 g of amberlite resin and stir 5 min. Filter through a 0.2 uM filter and degas at 25 mg Hg for 5 min. Transfer to graduate cylinder and add 10 ml 10x TBE, bringing volume to 100 ml with d.i. water.

4. We have recently started using Burst-Pack from Owl Scientific, which is an acrylamide premix including the buffer and catalysts, for our fluorescent gel work and are very pleased with the quality and reproducibility. This would be an option for the silver staining work as well. The burst-pack's eliminate the c hemical weighing and mixing, deionizing, filtering and degassing steps.

IV. GEL POURING:

1. Immediately prior to pouring, add 500 μl 10% ammonium persulfate (0.1 g + 1 ml d.i. water) to the acrylamide mix in a beaker, gently mixing well. Then add 50 μl TEMED and mix. Polymerization will not start until TEMED has been added. Do not mix the catalysts together before adding to the polyacrylamide solution--this will inhibit polymerization.

2. We use the Otter adjustable gel caster (OWL Scientific, Inc.) for pouring gels. In this system the gel is poured horizontally with the top plate sliding over the bottom plate, without the use of tape, grease or a bottom spacer.

a. Place the larger plate onto caster so that it abuts the end wall of the caster. Moisten spacers with water and place them flush to the edge of glass against the caster wall.

b. Place the top plate (notched or offset) so that its top edge overlaps the bottom edge of the lower plate by 3-4 cm. Using a 60 ml syringe, slowly dispense the gel solution between the plates, allowing the solution to flow by capillary action. Gently slide the top plate across the bottom plate while dispensing the gel solution along the leading edge of the top plate. If any bubbles form while pouring, try sliding the top plate back to uncover the bubble, then proceed. A more effective method is to drag out the bubbles with a plastic hook (free by request from Promega Corp.)

3. Once the gel is poured, insert the flat edge of a sharktooth comb (or a casting comb) into the top of the gel to the depth desired for the wells. Place 2-3 clamps along the sides and top to keep plates in tight contact with the spacers and comb while the gel is polymerizing.

4. Allow the gel to polymerize at room temperature (RT) for 1 hour. Gel can be stored at RT over night if steps are taken to prevent it from drying out. To do this, place paper towels dampened with running buffer over the top (remove clamps but leave comb in place) and bottom edges of the gel mold and wrap with plastic wrap. Do not store the gel under buffer.

V. SAMPLE PREPARATION:

Notes: Heat samples immediately prior to loading. Keep the loading dye fresh. Use SSRP loading dye that is less than 2 weeks old. The deionized formamide used in making the loading dye should be less than one month old.

1. Denature the sample DNA by adding 1 volume (10 ul) of fresh SSRP loading dye (10 mM NaOH, 95% formamide, 0.05% bromophenol blue, 0.05% xylene cyanol) to 1 volume of PCR sample in a microtiter plate. Mix well and heat to 95℃ for 2 min. Place on ice. 2. Molecular weight standards are PGEM (Promega) and Poly-dA (Pharmacia # 27-7836-01) sonicated to produce a 1 bp ladder. PGEM is loaded in a well separate from poly A. We do not use poly A for fingerprinting gels. Using a 144-well, microtiter 4X offset comb, load 3 μl of the mix:

3.4 μl 1X Perkin Elmer II PCR buffer

8.6 μl of 30 ng / μl PGEM

12 μl of SSRP buffer

heat 95o C for 2 min., ice

and

5 μl 1X PE II buffer

2.5 μl of 400 ng / μl of sonicated Poly-dA

7.5 μl SSRP bufferVI. ELECTROPHORESIS:

Note: Adding sodium acetate to the bottom reservoir during electrophoresis (Sheen and Seed, 1988, Biotechniques 6:942-944) produces the same effect as running wedge shaped gels or adding a gradient to the gels themselves (Biggin et. al., 1983, PNAS 80:3963-3965). In all three methods, the mobility of small DNA fragments is retarded as they approach the bottom of the gel. The sodium acetate method is simpler than the other methods.

1. Remove clamps. Clean excess polyacrylamide and urea from the top of plate assembly with d.i. water. Pull the comb out of the mold slowly and evenly, cleaning out the comb area with d.i.water or buffer.

2. Add reservoir buffers to the apparatus. The top reservoir buffer is 1X TBE. The bottom buffer reservoir is 2/3X TBE, 1 M sodium acetate. Make 1500 ml bottom buffer for each gel (100 ml 10x TBE, 900 ml d.i. water, 500 ml 3 M NaAcetate).

3. Pre-electrophorese for 5-10 min to warm the plate so that the comb will easily slide in place. Clean out comb area with buffer. To prevent possible well to well leakage, apply a very light coating of celloseal to the outside surface of the comb, prior to insertion. Place plate assembly in gel box and clamp.

4. For microtiter format, a hamilton 8 or 12 channel syringe loading device for loading the gels is recommended. Clean out each group of wells immediately prior to loading with the multichannel hamilton syringe. Run the gel at 50℃ constant temperature and 100 watts limiting power for about 1.5 - 3 hours, depending on size of amplification product. Constant temperature can be maintained with the temperature probe option of the BioRad Power/Pak 3000 power supply.

VII. GEL FIXING, STAINING AND COLOR DEVELOPMENT:

Notes and tips: -This procedure is adapted from the Promega Silver Sequence protocol. -Use highest quality reagents. -The fixer, stain and developer are made with 18 mega-ohm water (deionized= d.i.). -The washes are done with distilled water. -We use pre-measured sodium thiosulfate (-20℃) and silver nitrate (RT), stored in microtubes. -All incubations and washes are performed at room temperature, but the developer solution must be pre-chilled to 4-10℃ to minimize brown background. -It is important to keep the stop solution at 4-10℃ as well, for the same reason. -Solutions containing formaldehyde should be handled in a fume hood. The formaldehyde is aliquoted (RT) -Wear gloves throughout the procedure. - Wearing an apron will prevent silver stains on clothing. -Rocking platforms are better than orbital shakers to achieve even gel staining.

1. Freshly prepare staining and developer solutions while gel is running. Add formaldehyde, silver nitrate and sodium thiosulfate to solutions immediately prior to use. To prepare staining solution, combine 2 L d.i. water, 2 g silver nitrate and 3 ml 37% formaldehyde. To prepare developing solution, combine 2 L chilled d.i. water, 60 g sodium carbonate, 3 ml 37% formaldehyde and 4 mg sodium thiosulfate. Chill developer to 4-10℃.

2. Remove gel assemble from rig, remove side spacers and carefully separate glass plates with a spatula. The gel will remain attached to the plate coated with bind silane. Cut off the top corner of the high numbered end of gel for orientation.

3. Place gel and plate in a shallow plastic tray containing sufficient fresh fix/stop solution (~2 liter 7.5% glacial acetic acid) and gently agitate for 20 minutes or until tracking dye completely disappears. We sometimes use a multiple gel holder from Promega for processing several gels at a time. Two gels require the same volume of solution as one. For three gels, solution volumes are increased by 50%.

Note: For overnight storage, the gel can be fixed as above, rinsed with d.i. water and stored in fresh fixer without agitation.

4. Rinse the gel 3 times for 2 minutes each with distilled water with gentle agitation.

5. Add staining solution to gel and gently agitate for 30 minutes, decant and precipitate silver. The silver in the used staining solution is precipitated with NaCl (10 g /2 L) for recycling.

6. Rinse gel with distilled water for 5 seconds (from time gel is placed in distilled water to time into developer, i.e. a quick dip). Longer rinses result in weaker signal. If the rinse goes too long, repeat step 5 with the staining solution.

7. Transfer the gel to 800 mls of chilled developing solution and gently agitate with a rocking motion until the bands first become visible, usually within in a few minutes. Decant the used developer, add the remaining chilled developer and continue to gently agitate until the bands have reached desired intensity. Over-development will result in a brown background with low contrast bands rather than a pencil gray background with sharp bands.

8. To terminate the developing reaction, add an equal volume of chilled, fix/stop solution and incubate with gentle shaking for 5 minutes. Lift up plate to ensure that the neutralizing solution comes in contact with developer underneath the plate. As soon as the bubbling stops, rinse the gel thoroughly in d.i. water. Neutralizing too long will bleach out the bands.

9. Air dry the gel. Photographic contact prints can be made with APC film (Promega) for permanent record. (Develop APC film 3 min in X-ray film developer, wash 1 min., fix 3 min, wash 10 min. Processing temperature must be 20℃ or cooler, warmer temperatures cause the film to yellow.) A flatbed scanner can be used to capture digital images of the gel.

10. To dispose of gel, re-hydrate for a minimum of 10 min. in water. Scrape off the gel with a plastic scraper. Wash plates in alconox detergent, using sponges dedicated for each type of coating, rinse in d.i.water.

11. We remove the silanizing treatment from the plates every time they are used, and as needed for the acrylease coated plate (~every 3-4 times). To do this, soak the plate in 2M NaOH solution 1 hour and wash as above.

Our supplier and protocol for making the Poly A ladder:

1.  Polydeoxyadenylic Acid #27-7836-01 (5 U) from Pharmacia.

2.  5U / (A260/mg) to give you mg.  Add TE for a stock concentration of 400 ng/ul.

a. Place in 0.5 ml tube in ice bath (small beaker) and sonicate 30 sec

at 60% power (using a 3mm microtip with a 50W Vibra-Cell sonicator)

b. Clean tip by sonicating in d.i. water 5 sec before starting.

c. Wear ear protection

d. Store at -20℃