About ZR small-RNA™ PAGE Recovery Kit
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 Fast-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.
| Format | Spin Column |
|---|---|
| Processing Time | 45 min |
| Equipment | Microcentrifuge, 37 to 65ºC heat source, dry ice or -80ºC freezer. |
| Sample Sources | Single- or double-stranded RNA fragments (17-200 nucleotides) resolved in polyacrylamide gels (tested up to 25% (w/v) polyacrylamide) stained with ethidium bromide or ssRNA-specific dyes (e.g. GelStar®). |
| RNA Purity | High quality RNA (A260/A280>1.8, A260/A230>1.8) suitable for all downstream RNA-based manipulations. |
| RNA Recovery | The recovery rate for fragments 17 to 28 nucleotides is ≥50 %. Total binding capacity of the supplied Zymo-Spin IC™ Columns is ≥5 µg. |
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Recovery and ligation of single-stranded RNA oligonucleotides. In the image above, the RNA fragments were recovered from a 17.5% (w/v) native polyacrylamide gel using the ZR small-RNA™ PAGE Recovery Kit. All fragments shown were resolved in a native PAGE gel following ligation. T4 polynucleotide kinase and T4 RNA ligase I (New England Biolabs) were used for the phosphorylation and subsequent ligation of the ssRNA samples. Ligated RNAs are circled in yellow. RNA in the gel was visualized with GelStar® Stain (Lonza)
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| Step 1 | Excise an RNA fragment from a PAGE gel and transfer the slice into a Zymo-Spin™ IV Column in a Collection Tube. |
|---|---|
| Step 2 | Crush the gel slice with a Squisher™-Single against the side of the column. Add 400 µl RNA Recovery Buffer directly into the column. Cap the column and incubate at 65oC for 15 minutes. |
| Step 3 | Quick freeze the samples on dry ice or in a -80ºC freezer for 5 minutes, then transfer columns back into 65oC for 5 minutes to thaw. |
| Step 4 | Snap off the Zymo-Spin™ IV Column tip and place the column back into a Collection Tube. Centrifuge at ≥1,500 × g for 30 seconds. Save the flow-through. |
| Step 5 | Transfer the flow-through from the Step 4 to a Zymo-Spin™ IIIC Column in a Collection Tube and centrifuge at ≥1,500 × g for 30 seconds. Save the flow-through. |
| Step 6 | Add 2 volumes of RNA MAX Buffer to the flow-through from Step 5 and mix well. |
| Step 7 | Transfer the mixture to a Zymo-Spin™ IC Column in a Collection Tube. Centrifuge at ≥12,000 × g for 30 seconds. Discard the flow-through and place the Zymo-Spin™ IC Column back into the Collection Tube. |
| Step 8 | Add 400 µl RNA Prep Buffer to the column. Centrifuge at ≥12,000 × g for 1 minute. Discard the flow-through and place the Zymo-Spin™ IC Column back into the Collection Tube. |
| Step 9 | Add 800 µl RNA Wash Buffer to the column. Centrifuge at ≥12,000 × g for 30 seconds. Discard the flow-through and place the Zymo-Spin™ IC Column back into the Collection Tube. |
| Step 10 | Repeat Step 9 with 400 µl RNA Wash Buffer. |
| Step 11 | Centrifuge the Zymo-Spin™ IC Column at ≥12,000 × g for 2 minutes in an empty Collection Tube to ensure complete removal of the wash buffer. |
| Step 12 | Place the Zymo-Spin™ IC Column into a provided DNase/RNase-Free Tube. Add 6-15 µl of the provided DNase/RNase-Free Water directly to the column matrix and let stand at room temperature for 1 minute. |
| Step 13 | Centrifuge the Zymo-Spin™ IC Column at 10,000 × g for 1 minute to elute RNA. Recovered RNA can be used immediately or stored at ≤-70 oC. |
