When optimizing the experimental conditions, the PCR products should be separated on gels containing glycerol and without glycerol at the same time and the results of the radioactive autoradiography should be compared. Pre-experimental results using both gels to analyze the same sample can be obtained within 24h. Once the experimental conditions are determined, the gel type preferred for a particular experiment can be applied to the analysis of all samples of that locus. This experiment was derived from PCR Laboratory Guide (Second Edition) by Seed Kang and Qu Lijia.
Operation method
Gel electrophoresis experiment
Materials and Instruments
Acrylamide Bisacrylamide Storage Solution Ammonium Persulfate Ethanol Sampling (Termination) Buffer Formamide EDTA Bromophenol Blue Xylene Nitrile Enzyme and Enzyme Buffer PCR Products (Radioactive) Gels Culture Media Move I. Materials For more product details, please visit Aladdin Scientific website.
ZapCap Filters 108-well deep well combs Chromatography paper Samplers Acrylic Sheets Film Dark Boxes Gel Blotting Films Geiger Counters Gel Drying Systems Gel Film S2 Sequencers Paraffin Sealing Films Pipettes Razor Blades Reagent Reservoirs Thermal Cyclers (PCR Instruments)
1. Buffers, solutions and reagents
40% acrylamide/diacrylamide storage solution, 37.5:1 (BIORAD)
10% Ammonium persulfate
Dispense 100 ml and store in 10 ml portions at -20°C. Screw-cap borosilicate scintillation vials make good storage containers.
Ethanol, 95 percent
2X Sampling (Termination) Buffer
95% Formamide
20 mmol/LEDTA
0.05% Bromophenol Blue (Sigma-AldrichB5525)
0.05% xylenecyanol (Sigma-AldrichX4126)
Dispense 300 ml, then divide into small portions into 15 ml conical tubes and store at -20°C. For ease of use with multichannel pipettes. To facilitate pipetting, transfer a small portion to a sterilized reagent reservoir or tank (Eppendorf or Apogent Discoveries).
10XTBE Electrophoresis Buffer
Tris base 18 g
Boric acid 55 g
EDTA 9.3 g
ddH2O to 1L
N,N,N',N'-Tetramethylethylenediamine (TEMED)
Safety Coat Non Toxic Glass Plate Coating (Caution: Flammable)
2. Enzyme and enzyme buffer
3. nucleic acids and oligonucleotides
PCR products from Scheme 2 (radioactive)
4. gels
(1) SSCP gel mixture with 10% glycerol (final %C = 2.67%)
10XTBE 50 ml
40% acrylamide (37.5:1) 75 ml
ddH2O 325 ml
Clarify by filtration through a 0.45um ZapCap port, add 50 ml of ultrapure grade glycerol and store at 4°C away from light (wrap solution bottle in aluminum foil).
(2) Glycerol-free SSCP gel mixture (final %C = 2.67%)
10XTBE 50 ml
40% Rienamide (37.5:1) 75 ml
ddH2O 375 ml
Clarify by filtration with 0.45um ZapCap and store at 4°C protected from light (wrap solution bottle in aluminum foil).
5. Culture medium
6. Specialized equipment
0.45um ZapCap filter (SchleicherSchuell)
108-welldeep-well comb (108-welldeep-wellcomb)
This is a square-well shaped comb that is custom machined at The Gel Company in San Francisco, CA (www.gelcompany.com, 1-800-256-8596). This type of comb is preferable to the shark tooth comb (shark, stoothcomb) due to the fact that it can form distinct individual spiking grooves. The leakage or mixing of samples between adjacent spiking slots that can result from shark tooth combs does not occur with square donut shaped combs, and such leakage or mixing can interfere with the interpretation of the data.
3 MM46 cmX57 cm Chromatography Paper (Whatman)
8-channel injectable gel sampler (Hamilton)
It should be noted that it is more economical to purchase the Model 84501 12-Channel Sampler. By removing 2 injection tips at each end, for a total of 4 injection tips, a free section is automatically left for maintenance. The syringes need to be replaced periodically, are more expensive to order individually, and they are often out of stock, which can delay experiments.
Acrylic Sheet
Reagent bottle, 125 ml
Conical Tube, 15 ml
Film cassette with sensitizing screen
GB002 Gel Blotting Film (Schleicher&Schuell)
Geiger counter (a radiometric energy meter)
Gel drying system (BioRadModel583withHydrotechpump or similar)
Kimwipe (- a thin soft absorbent paper, trade name) paper towels or other dust free paper
KodakXAR514X17 Film
Large clips
Sequencer Model S2 (previously supplied by LifeTechnologies, now supplied by WhatmanBioMetra) with 0.4 mm spacer (spacer) and standard beveled glass plate
Parafilm paraffin sealing film
Pipette, 50 ml
Power supply.
Razor blades
Reagent reservoir (Eppendorf or ApogentDiscoveries)
SafetyCoatNonToxicCoating (JTBaker) (note: flammable)
Cling Film (Saran Wrap, SamnWrap)
Syringe
Thermaladhesivesealingfilm (FisherScientific), used to seal the 96-well plate when the sample is denatured
Thermocycler (PCR instrument), or heat block
Methods
1. Installation of the glass plate
(1) Prior to installation, small glass panels should be treated with a silanizing agent such as Safety Coat Non Toxic Glass Plate Coating, following the manufacturer's instructions.
(2) Clean the glass plate with a soft sponge and a non-abrasive detergent. The glass plate must be very clean and free of paper to minimize the formation of air bubbles.
(3) Rinse the slide with ddH20 and scrub with a large Kimwipe paper towel. Rinse with 95% ethanol and dry most with Kimwipe tissue.
The water should condense into small droplets on the small slide.
(4) Place a clean gasket on the surface of the large breakout board and place the small breakout board on top of it, making sure that the foam piece of the gasket is flush with the top of the small breakout board to avoid leakage.
(5) Clamp the panes together along the edges (about 4 clips per side). The inside of the clips should not extend beyond the edge of the gasket.
(6) Place the slide unit horizontally, but slightly elevated (polystyrene foam tube holders work well). Take out the comb and other materials for gel preparation.
2. Gel instillation
(1) Transfer 75 ml of gel mixture to a 125 ml bottle. Add 37ul of TEMED, seal the membrane with Parafilm paraffin and shake vigorously. Add 705ul of 10% ammonium persulfate: seal with Parafilm paraffin sealing film and shake vigorously. Remove the air with its vacuole.
(2) Fill a 50 ml pipette with the gel solution, tilt the clamped slide (the top of the slide is higher), and move the pipette from one end of the top of the slide to the other, so that the gel solution slowly and evenly disperses between the two broken plates. When the gel reaches the bottom, flatten the slide. Quickly insert the teeth of the gel comb between the slide plates. The top of the spiking groove should be flush with the edge of the glass plate.
(3) Clamp an additional clip on the broken plate at the edge of the comb.
(4) Allow the gel to polymerize for at least 40 min.
3. Gel rigging
(1) Remove all clamps.
(2) Gently loosen with a razor blade underneath the comb, then remove the comb slowly and evenly.
(3) Place the broken plate in the gel box and pour 1XTBE full in the buffer chamber.
(4) Rinse the spiking tank by drawing up 1XTBE buffer with a syringe. If the spiking slot is bent, use a flat pipette tip to straighten the teeth between the spiking slots.
(5) A pre-run of the gel is not necessary; proceed with sample preparation and loading.
4. Sample Preparation and Electrophoresis
(1) If the PCR product from Protocol 2 has been frozen, it is thawed and then centrifuged briefly to remove the solution from the cap and tube walls.
The PCR product is radioactive and requires appropriate protection when handled.
(2) Remove the lid carefully. Determine the radioactivity of the gloves with a Geiger counter and replace the gloves if necessary.
Removal of the lids by wrapping in Kimwipe tissue reduces radioactive contamination of the gloves.
(3) Add an equal volume of (12.5ul) 2X Termination Buffer to all samples using a multichannel pipette. Blow and mix to cover the slide with heat sealing film.
(4) Denature the samples for 5 min at 95°C on a heat block or PCR instrument, then quickly place on ice. Remove and discard the heat-sealing film, change gloves, and top up the sample with a multichannel injectable gel sampler. The remaining sample can be stored at -20°C.
(5) Connect the gel cassette to the power supply with the power cord and turn on the power.
Note: High voltage.
Start by electrophoresing the sample into the gel at about 40W. If a gel containing glycerin is used, reduce the power to 8W and electrophoresis for about 17 h (overnight). If a glycerol-free gel is used, reduce the power to 25 W and electrophoresis for about 6 h. Electrophoresis is performed until xylene cyanide (the dye in the sample buffer) has run through nearly 2/3 of the gel.
(6) When electrophoresis is complete, turn off the power and disconnect the cord from the gel electrophoresis cartridge. Remove the gel from the gel electrophoresis cartridge and lay the small slide flat face up. Slide out the two spacers and use a razor blade to pry off the top slide.
(7) Place a Whatman3 MM filter paper on top of the gel and press it gently without rubbing it. Place a piece of blotting film on top and press gently. Hold the slide underneath with one hand, place the other hand on the blotting film, and invert the device. Pry the slide off the surface of the gel. Cut off the excess paper around the gel and cover with plastic wrap.
(8) Dry the gel at 80°C for about 45 min, transfer to a film dark box, and expose the X-ray film (exposure time can be determined when optimizing the experiment). The negative is developed, the sample labeled, and analyzed.
Be sure to check all work areas and instruments with a Geiger counter throughout and after the experiment. Cleaning and decontamination are necessary.