Member since: 2021
Organization: Stanford University
Great kit - works for 50-350cubic nm RNAs at high concentrations (routinely get 100s pmols).
Application Area: RNA size selection and purification
"I use this product to purify large amounts of 50-350nt RNA using denaturing PAGE. This kit works far better than the alternative "crush and soak" method - both with yield (>2x greater) and rate (1 evening possible vs a mandatory O/N step). However, it did take some trial and improvement to optimize this kit for larger RNAs / larger loads. Firstly, one column can comfortably fit about 30,000 cubic mm of gel. This gel should ideally be at 10% polyacrylamide (29:1) though 8% works, lower concentrations lead to clogging during protocol and worsen yield. The step timing is all off - Zymo is clearly trying to market this as a fast protocol: it's faster than the alternative, but it is not as fast as they say. Freeze the crushed and soaked gel at -80C prior to the heat treatment (do the -80C step after the treatment too) - it's fine to leave the samples overnight in the -80C. After the heat treatment, spin at max speed (~20k Gs) for 5-10mins - this will shred most of the gel through into the collection tube. Next spin through the second tube at 5k Gs for 5mins - do not spin this too quickly or you will shred the gel through the column and this will lead to clogging the next column. If when you add the RNA MAX buffer and you see a white precipitate forming, spin this out and try your best to not transfer this to the last column - this is caused by gel particulates not being filtered out by the second column (spin too fast). Then follow the protocol as written. This kit also works for PAGE purification of cDNA / ssDNA. Suggestions to improve: 1) Many of the steps require transfer of volumes from collection tubes. these tubes have hemispherical bottoms (vs the standard conical bottoms of a microcentrifuge tube) and this makes it hard to transfer all of the liquid. Please change these tubes to conical collection tubes. 2) There's a step where you add "2x the eluted buffer" please place graduation makers on the collection tube to help estimate this."
The ZR small-RNA PAGE Recovery Kit provides an easy and efficient method for the extraction of high quality small RNAs from polyacrylamide gels (native and/or denatured). The ZR small-RNA™ PAGE Recovery Kit is a refinement of the "crush and soak" method that incorporates a unique buffer system together with Zymo-Spin column technologies for improved recovery and added convenience. The recovered RNA can be concentrated into volumes as small as 6 µl, making it ideal for many downstream enzymatic reactions and manipulations.