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- How do I prepare a denaturing polyacrylamide gel?
- 2. Prepare a denaturing polyacrylamide gel as described in Polyacrylamide Gel Electrophoresis of RNA (Rio et al. 2010). Set up the gel in the gel box, add TBE electrophoresis buffer (diluted to 1×) to the upper and lower reservoirs, and prerun the gel for 15–45 min at a maximum of 1500 V/45 mA.
- What is polyacrylamide gel electrophoresis (PAGE)?
- Preparative polyacrylamide gel electrophoresis (PAGE) is a powerful tool for purifying RNA samples. Denaturing PAGE allows separation of nucleic acids that differ by a single nucleotide in length.
- What are the elution conditions for a polyacrylamide gel?
- The elution conditions can vary greatly depending on the electroelution apparatus, the acrylamide percentage of the gel, the membranes, and the buffer used. A good starting point is to conduct the electrophoresis in 1× TBE at 100V for 2h. This should be sufficient to elute ~100 nucleotide long RNA molecules from a 4% polyacrylamide gel.
- How is RNA extracted from a polyacrylamide gel?
- Overview RNA will be extracted from the gel using the ‘crush and soak’ method (see also Purification of DNA Oligos by Denaturing Polyacrylamide Gel Electrophoresis (PAGE)). 8.2. Duration
