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.
|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.|
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)
Excise an RNA fragment from a PAGE gel and transfer the slice into a Zymo-Spin™ IV Column in a Collection Tube.
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.
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.
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.
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.
Add 2 volumes of RNA MAX Buffer to the flow-through from Step 5 and mix well.
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.
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.
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.
Repeat Step 9 with 400 µl RNA Wash Buffer.
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.
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.
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.