Tri borat

Stellwagen, NC, Apparent Pore Size of Polyacrylamide Gels Comparison of Gels Cast and Run in Tris-acetate-EDTA and Tris-borate-EDTA Buffers, Electrophoresis 19, 1542, 1998. Stellwagen, NC Gelfi, C Righetti, PG, The Free Solution Mobility of DNA, Biopolymers 42, 687, 1997. 

FIGURE 18.2 Capillary gel electrophoresis separation of an octylphenol ethoxylate sulfate (with an ethylene oxide chain length from 1 to 8). Run conditions pH 8.3 (100 mM tris-borate, 7 M urea) 50 pm x 75 cm J W polyacrylamide gel capillary (PAGE-5, 5%T, and 5%C) run at 20 kV with a 5kV injection for 5 s UV detection at 260nm. 

Fig. 17.11. Bottom CGE separation of components of poly U (sigma) in 25% pluronic F127. Top Note the resolution of two contaminants between each of the oligonucleotides from about 15 to 27 nucleotides long in this expanded section of the bottom electropherogram. Electrophoresis was performed in 25% pluronic F127 in tris-borate-EDTA buffer (90 mM tris, 90 mM boric acid, 2 mM Na EDTA, pH 8.3.) (25°C, 500 V cm-1, effective column length 30 cm). Reprinted with permission from Ref. [82],...

Other buffers that have been used for continuous, native electrophoresis are Tris-glycine (pH range 8.3 to 9.5),19 Tris-borate (pH range 8.3 to 9.3),26 and Tris-acetate (pH range 7.2 to 8.5).27 Borate ions26 can form complexes with some sugars and can therefore influence resolution of some glycoproteins. 

Tris-sulfate/Tris-borate, Tris-formate/Tris-borate, and Tris-citrate/Tris-borate have been advocated as electrophoresis buffers.5 For basic proteins, a low-pH alanine-acetate system28 is often used. 

List of Abbreviations PCR, polymerase chain reaction RT-PCR, reverse transcription polymerase chain reaction DNA, deoxyribonucleic acid RNA, ribonucleic acid RNase, ribonuclease mRNA, messenger RNA GABAa, y-aminobutyric acid type A cRNA, copy RNA dNTPs, deoxy nucleoside triphosphates MMLV, Mouse Moloney murine leukemia vims RT, reverse transcriptase bp, base pair Tm, melting temperature DEPC, diethylpyrocarbonate OD, optical density mL, milliliter SA-PMPs, streptavidin paramagnetic particles dT, deoxy thymidine DTT, dithiothreitol DNase, deoxyribonuclease RNasin, ribonuclease inhibitor UV, ultraviolet TBE, Tris-borate, 1 mM EDTA EDTA, ethylenediaminetetraacetic acid Buffer RET, guanidium thiocyanate lysis buffer PBS, phosphate buffered saline NT2, Ntera 2 neural progenitor cells... 

Tris-borate-EDTA (TBE) buffer (lOx solution) 0.89 M TRIZMA base, 0.89 M H3BO3,20 mM EDTA. Per liter 108 g TRIZMA base, 55 g boric acid, 40 ml 0.5 M ethylenedi-aminetetraacetate (EDTA) solution. 

Standard research-grade agarose is usually sufficient, but special agaroses may be used for specific applications (e.g. high-resolution gels). The electrophoresis buffer is usually prepared and stored as a 10 x concentrated stock. The most commonly used buffer is Tris-borate-EDTA (TBE), or alternatively, one may use Tris-acetate-EDTA (TAE) buffer ... 

Buffers appropriate for electrophoresis gels include Tris-glycine, Tris-acetate, Tris-phosphate, and Tris-borate at concentrations of about 0.05 M. 

Nucleic acids can be visualized by ethidium bromide staining, UV shadowing, or phosphorimaging of radioactive samples. A sterile scalpel should be used to excise the separated product which can then be eluted by electrophoresis (1 x Tris-borate, pH 8.3) into a 30kDa molecular weight cutoff (MWCO) filter (Millipore). The product is concentrated by centrifugation, dialyzed, and resuspended in the buffer of choice. The yield of nucleic acid is typically 75 %, and ethanol precipitation is not needed. 

Schwinefus, J. J. and Bloomfield, V.A. (2000) The greater negative charge density of DNA in tris-borate buffer does not enhance DNA condensation by multivalent cations. Biopolymers, 54, 572-577. 

The species to be compared are loaded in 6% Ficoll (w/v) onto an 8% or 10% polyacrylamide gel with 29 1 monomer bisacrylamide. We generally use 90 mM Tris—borate (pH 8.3) buffer containing either EDTA or added metal salts. Electrophoresis is typically performed for 1—2 days at 5 V/cm at room temperature. When salts are included in the electrophoresis buffer, this must be recirculated between the cathodic and anodic reservoirs at a rate of 1 L/h. This may require a little modification of commercial gel electrophoresis apparatus. Our RNA species generally include at least one strand that is radioactively [5/-32P] -labeled. When the electrophoresis is complete the gel is dried onto Whatman 3MM paper and exposed to storage phosphor plates at 4 °C followed by phosphorimaging to provide a gel image. 

Figure 7.3 Analysis of the 4H four-way RNA junction of the human U1 snRNA by comparative gel electrophoresis (Duckett et al., 1995). The central sequence of the junction is shown. The A G pair at the center was retained in this analysis, although changing it to a Watson—Crick pair did not alter the global shape of the junction. The six long—short species can be considered to be derived from a junction with four arms of 40 bp. The central 20 bp comprises RNA, and the outer arms are DNA. The junction species were electrophoresed in an 8% polyacrylamide gel, in 90 mM Tris—borate (pH 8.3) and 1 mM Mg2+. The mobility pattern of the six species is slow, slow, fast, fast, slow, slow. The simplest interpretation (shown on the right-hand side) is that of a stacked structure based on A on D and B on C coaxial stacking, with the axes nearly perpendicular. The pattern would also be consistent with a rapid exchange between nearly equal populations of parallel and antiparallel forms. However, a recent crystal structure has found a perpendicular stacked structure for this RNA junction (Pomeranz-Krummel et al., 2009).

Figure 7 A Analysis of the 2HSj2HS2 four-way junction of the HCV IRES by comparative gel electrophoresis. The sequence of the junction around the point of strand exchange is shown. Comparative gel electrophoresis in a 10% polyacrylamide gel was performed in the presence of 90 mM Tris-borate (pH 8.3), 1 mM Mg2+, using the six long-short arm species, where arms were extended with DNA sections as before. The observed pattern of mobilities is interpreted in terms of a rapid exchange between approximately equal populations of parallel and antiparallel conformations as shown, with strand polarities indicated for clarity.

Figure 7.5 Analysis of the two three-way RNA junctions of the VS ribozyme by comparative gel electrophoresis. The secondary structure of the VS ribozyme is shown, with the sequences of the two component three-way junctions. Each was analyzed in isolation by comparative gel electrophoresis, comparing the mobilities of the three long-short arm species. As before, these species have a central core of RNA that is extended with DNA sections. The junction species were electrophoresed in 10% polyacrylamide gels in the presence of 90 mM Tris—borate (pH 8.3) with 3 (junction III—IV—V) or 5 (junction II—III—VI) mM Mg2. The structural interpretations of both sets of data are shown. Both junctions undergo coaxial stacking of two arms, with die third directed laterally.

The donor—acceptor-labeled RNA is then constructed by hybridization. The required stoichiometric combinations of fluorophore-labeled and unlabeled strands are mixed together in 90 mM Tris—borate (pH 8.3), 25 mM NaCl for 10 min. They are then placed at 80 °C, followed by slow cooling over several hours. The doubly labeled species are purified by electrophoresis in a polyacrylamide gel under nondenaturing conditions at 4 °C at 150 V in 90 mM Tris—borate (pH 8.3), 25 mM NaCl with recirculation at >1 L/h. The fluorescent species are recovered by band excision and electroelution. 

Buchmueller, K. L., and Weeks, K. M. (2004). Tris—borate is a poor counterion for RNA A cautionary tale for RNA folding studies. Nucleic Acids Res. 32, el84. 

Figure 8.21. Electropherograms of the Hae-III digest of FX-174-RF DNA using (a) an agarose slab gel (total running time was approximately 40 min), (b) a polyacrylamide-coated capillary, and (c) microchips on poly(methyl methacrylate) substrate. The separation buffer for both polyacrylamide-coated capillary and microchips was 1.5% HPMC in TBE buffer (100 mM Tris-borate and 5 mJf EDTA, pH 8.2) with 10 6 M of TO-PRO-3. (Reprinted with permission from Ref. 44.)...

Fig. 12. Experimental setup for the frequency comparison between the IS — 3S and 2S — 6S/D transitions (TiSa titanium sapphire laser, LBO lithium tri-borate crystal, BBO /3-barium borate crystal)...

Electrophorese a 5 m1 sample of the above digest in parallel with 4m1 of the untreated RFI solution on a 1% agarose gel in tris-borate buffer (lOOmM-Tris base, lOOmM-boric acid, 2 mM-EDTA). Dilute each sample into 15 m1 H20 and add 3m1 loading buffer (3% bromophenol blue in 0.1M-EDTA, pH 8.3, 50%... 

The thin-layer gel system used for resolving the different fragments produced by the chemical degradation method is essentially that of Sanger, Nicklen and Coulson (1977) as described in Chapter 4. An 8% polyacrylamide gel is usually used though for special purposes a 10 or 12% gel can give an improved resolution of the smallest fragments. For the standard 8% gel the polymerization mixture contains 7.6% (wt/vol) acrylamide, 0.4% (wt/vol) bisacrylamide, 50% (wt/vol) urea, 100 mM Tris-borate, pH 8.3, 2mM EDTA, 0.07% (wt/vol) ammonium persulphate and TEMED catalyst. This solution is poured or injected into a 0.4 x 200 x 400 mm mold to form a gel slab. 

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