Post Electrophoretic Analysis Articles
Preparing Denaturing DNA & RNA Gels
Sequencing gels are poured between two glass plates separated by spacers. The spacers are typically no more than 0.2mm in thickness. The extreme thinness of the gel allows air bubbles to be trapped in the gel during pouring. Such bubbles are very hard if not impossible to remove. As oxygen inhibits the polymerization process, even a small bubble can create a hole in the gel which is large enough to prevent the use of several lanes. Bubbles can be avoided by using scrupulously clean plates, by siliconizing one plate, and by vigilance during the casting process.
Sequencing Gel Separation Using SequaGel – UreaGel 6
- Clean glass plates thoroughly. Use a laboratory detergent and scrub with a gloved hand or a paper towel. Rinse thoroughly with tap water; observe the flow of water over the plate (Unclean areas often cause distortions in the flow). Wipe the plate with a glass cleaner and rinse thoroughly with distilled water. Rinse with ethanol and wipe dry.
- Apply Glass FreeTM to one glass plate to ensure the gel will release from one plate after electrophoresis. This also will reduce the tendency of the treated plate to trap air bubbles.
- Assemble the cassette. Use the clamps provided with the apparatus, if available. If clamps are not provided, use binder clips, one every 20 cm along both edges. Place the cassette on a level surface which is shorter than the plates, so that the top and bottom of the gel extend beyond the surface. A thick book works well.
- Add 80ml of SequaGel 6 Monomer Concentrate and 20ml SequaGel Complete Buffer to a thick-walled Erlenmeyer flask.
- If desired the solution may be degassed for two minutes. Apply a vacuum (an aspirator is sufficient) while stirring or shaking. Bring to room temperature before polymerization.
- Add 800 µl FRESHLY PREPARED 10% ammonium persulfate, swirl gently to mix, and cast the gel.
TO CAST: with the gel lying flat, pour gel solution into the gap between the short and long plates, along the entire width of the gel. Capillary action will draw the solution into the cassette. Pour solution steadily to keep the top gap filled, and tilt the cassette 10-20° to encourage flow toward the bottom. Tap the cassette with a rubber stopper rapidly to keep bubbles from being trapped. If the plates are clean, the solution will flow from top to bottom smoothly in a line across the cassette. When the solution has reached the end of the cassette, return the gel to a level position, and insert the comb, flat edge first. Allow the gel to polymerize one to two hours.
- After two hours of polymerization wrap each end of the gel cassette with clear plastic wrap. NOTE: This is important to keep the ends of the gel from drying and to maintain sample well integrity. Appropriately wrapped gels may be stored for up to 48 hours at room temperature.
- Mount the gel in the running apparatus. Fill the upper chamber with buffer and remove the gel comb. Replace the comb with the teeth facing the gel, and insert until the teeth just penetrate the gel (no deeper than 1mm). Fill the lower chamber.
- Prerun the gel for 15-30 minutes before loading the samples, or as recommended for the apparatus used. For small (35ml, 20 x 40 x 0.04cm) gels use 30-35 watts. For large (70ml, 40 x 40 x 0.04cm) gels use 55-65 watts. The gel temperature should be between 45-50°C.
NEXT TOPIC: Run Conditions for Denaturing PAGE
- Using PAGE to Determine Nucleic Acid Molecular Weight
- SSCP Analysis
- Sanger Sequencing
- Sample Preparation for Native PAGE of DNA
- Sample Prep for Denaturing PAGE of DNA
- S1 Mapping
- Run Conditions in Denaturing PAGE
- RNA Mapping
- RNA Electrophoresis
- Ribonuclease Protection
- Restriction Digest Mapping
- Primer Extension
- Preparing Denaturing DNA & RNA Gels
- Preparation of Denaturing Agarose Gels
- Preparation of Agarose Gels
- Pouring Sequencing Gels
- PFGE and FIGE
- PCR Analysis: Yield and Kinetics
- PCR Analysis: An Examination
- Native PAGE of DNA
- Mobility Shift Assay
- Methylation & Uracil Interference Assays
- Maxam & Gilbert Sequencing
- Manual Sequencing
- In Gel Enzyme Reactions
- Heteroduplex Analysis
- Gel Preparation for Native PAGE of DNA
- Gel Electrophoresis of PCR Products
- DNase I Footprinting
- DNA/RNA Purification from PAGE Gels
- DNA/RNA Purification from Agarose Gels – Electroelution
- Differential Display
- Denaturing Polyacrylamide Gel Electrophoresis of DNA & RNA
- Conformational Analysis
- Automated Sequencers
- Analysis of DNA/Protein Interactions
- Agarose Gel Electrophoresis of DNA and RNA – Uses and Variations
- Agarose Gel Electrophoresis of DNA and RNA – An Introduction
- Sample Preparation
- Electrophoresis Buffers
- Blotting and Detection Buffers
- Gel Matrices
- Gel Accessories
- Ultra Pure Reagents