Gel purification

Full-length RNA molecules up to about 500 nucleotides can be purified by denaturing polyacrylamide gel electrophoresis. Following electrophoresis, RNA is localised in the gel by UV254-shadowing over Xerox paper, and a gel slice containing the full-length molecule is excised. The gel slice is crushed and continuously shaken at room temperature with equal volumes of 0.25 M sodium acetate (pH 6.0), 1 mM EDTA and phenol, followed by chloroform extraction and precipitation with ethanol (see below for further details). 

The necessary time of shaking will depend on the size of RNA, but overnight elution of reasonable amounts of large RNAs is feasible. 

Alternatively, large RNA molecules can be electroeluted from the gel slice into a dialysis bag containing TBE buffer by placing the dialysis bag perpendicularly to the voltage gradient in an electrophoresis chamber with TBE and electrophoresing at 10 V/cm for 1 h. 

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Columns packed with silica gel Purification 60 (230-400 mesh ASTM) hexane/ethyl acetate gradient Perkin-Elmer 1730FT1R Bruker WP 250 MHz using the solvent peak as a reference peak... 

Table 2 Optical Stability of Z-a-Amino Aldehydes and their Semi-carbazone Derivatives During Silica Gel Purification 11 ...

The HC and LC PCR products are pooled separately, phenol/chloroform-extracted, and ethanol-precipitated. Following resuspension and, where appropriate, gel purification of PCR products, the DNA is quantitated, before restriction enzyme digestion and cloning into thepComb3 vector. Gel purification (see below for methods) may be necessary when there are a high number of background bands in the PCR reaction. This tends to be more of a problem... 

Native polyacrylamide gel purification (Section 8.3.1.5) is required to isolate products of the appropriate size. 

Gel purification is a rapid and efficient way of isolating nucleic acids of the appropriate size from syntheses, PCR reactions, ligations, or tethered product-binding reactions. For preparative separation of random libraries (—150 bases) the following two types of polyacrylamide gels are used ... 

Treatment of acids 397 with me/a-chloroperbenzoic acid in a dichloro-methane solution after 2 h at room temperature and silica gel purification... 

Although agarose gel purification or commercial PCR clean-up kits can be used to process the DNA produced in these PCR reactions, we have found this to be unnecessary in most cases. However, if yields of DNA are very low, or substantial amounts of products other than the desired products are obtained from the reactions, purification of the DNA products may be necessary. 

Before gel purification, the percent of the mixture annealed (dsRNA substrate) should be determined. A small amount (usually 200 pmol by OD) of the crude unpurified substrate is end-labeled with 32P and electrophoresed in a 15% polyacrylamide gel. The gel is dried onto Whatman DEAE paper, and the radiolabeled RNA bands are visualized by autoradiography. Determine the amount of label in the dsRNA and ssRNA bands using a phosphorimager, or cut the bands out of the dried gel using the autoradiograph as a template and measure the amount of label incorporated into each band with a scintillation counter. Use this data to calculate the percent of the mixture that is dsRNA. This number is used to determine the yield of purified dsRNA. 

For gel purification, 20CM-00 pmol of 32P-labeled RNA crude mixture is mixed as a tracer with 40,000 pmol of unlabeled crude substrate. Measure the radioactivity of a small aliquot of the mixture in a scintillation counter to calculate the specific activity of the RNA. The dsRNA and ssRNA in the crude mix are separated by electrophoresis in a preparative 8% polyacrylamide gel. After visualization by autoradiography, the region of the acrylamide gel containing the dsRNA is excised with a razor blade and the RNA is extracted from the gel. After gel purification, the dsRNA substrate is suspended in buffer I, and the radioactivity of an aliquot is measured on the scintillation counter. From the specific activity of the dsRNA, the amount of gel-extracted substrate can be determined. This is called the cold substrate because the specific activity is lower than that of the hot substrate, which is freshly labeled with 33P for use in the assay. Since a small amount of the purified dsRNA is labeled with 32P, aliquots of gel-purified cold substrate should be stored in an acrylic (3-radiation storage container until they the radiation has decayed with time. 

The method in this section is used to label the crude, unpurified RNA mix-ture with 32P prior to gel purification. To label the gel-purified dsRNA with for assay use, substitute the [y-32P]-ATP with [y-33P]-ATP in the T4 PNK labeling reaction. 

The addition of glycogen to the ethanol precipitation step after labeling and gel purification increases the recovery of the precipitated RNA. After ethanol precipitation, the white RNA pellet is easily displaced from the bottom of the tube if the tube is jostled or allowed to stand. Remove the supernatant immediately after the microfuge centrifugation is completed. Take special care with the second pellet from the ethanol wash—it seems to stick less well to the tube and often floats loose. If this happens, use a pipet tip and carefully remove the supernatant without sucking up the pellet—do not use the aspirator. 

After gel purification, the dsRNA substrate should be validated using a gel-based assay, as well as the 96-well plate assay. The gel-based assay requires 32P-labeled substrate for autoradiography. When 32P-labeled substrate is used in the 96-well assay, care must be taken to use low amounts of radioactivity or to leave blank wells around the reactions owing to 32P cross talk between wells. 

Priming on M13 vectors resembles in vitro transcription but requires gel purification of probe, stability with duplex lower than with transcripts, DNA probes more stable than RNA probes... 

Fig. 7.15. Use of M13 for probes. Clones with antisense inserts (I) are labeled using an upstream primer whereas those with a sense insert (II) are labeled using a downstream primer followed by gel purification.

Chromatographic purification of individual SR-proteins is difficult due to their structural similarity. A SDS gel purification procedure involving a renaturation step has been published.11... 

Gel purification of amplified VH and VL genes may be necessary if many bands are observed from the PCR. 

Gel purification of amplified scFv gene may be necessary if many product bands are observed from the PCR. VL for and VH rev oligos are designed to introduce GlySer linker between VH and VL genes. The linker sequence is ... 

The amplified mutagenic DNA can be gel purified to remove the original template from mutagenized PCR product. Practical experience has shown contamination ofWT scFv pYDl plasmid has not been problematic due to minimal amount of intact plasmid and relatively low transformation frequency of yeast. However, gel purification should be used if PCR does not generate a discrete product band of appropriate size or one wants to eliminate all WT plasmid. 

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