Benchmarks Removal of Coomassie Blue Precipitates from Polyacrylamide Gels BioTechniques 21:820 (November 1996)
During staining of polyacrylamide gels, varying amounts of Coomassie blue may precipitate on the surface of the gels. This background obscures faint bands and is esthetically unappealing. The problem is worse when gels are stained for longer periods of time or when the stain has not been recently filtered. Rinsing a gel with methanol efficiently and quickly removes this background. Basically, at any time during destaining, gels are treated for 1 min in 100% methanol and returned to the destain or water. Gentle shaking is required. If the methanol rinse is done before excess stain is completely removed, it is easier to judge when destaining is complete (because of the lower background). During the treatment, gels may turn opaque, although this does not appear to harm the gel.
For illustration, a 0.75-mm-thick sodium dodecyl sulfate 10% polyacrylamide gel (1) was stained with 0.25% Coomassie Brilliant Blue R®-250 (BioRad, Hercules, CA. USA) in 25% isopropanol and 10% acetic acid for 24 h and then destained in 7% acetic acid with four changes of destain over 24 h (2). The gel was cut in half. One half was kept in destain, while the other was rinsed in methanol for 1 min (Figure 1), as described above. Ethanol and acetone removed precipitated Coomassie stain at about the same rate as methanol. Isopropanol was slightly less effective. Neither 0.75-mm gels nor 1.5-mm gels (10% polyacrylamide) appeared to be harmed by 5min treatments with 100% methanol, although the stain came off the gel surface in less than one minute. Protracted incubation will remove stain from protein bands. Also, 0.75-mm 5% and 20% polyacrylamide gels appeared unharmed by the methanol treatment. REFERENCES 1.Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of the bacteriophage T4. Nature 227:680-685. 2.Promega Corporation. Promega Protocols and Applications Guide. 1991. 2nd ed., p. 255. David E. Titus (Ed.). Madison, WI.
This work was supported by the U.S. Naval Medical Research and Development Command, Bethesda, MD, work unit No. B690.00101.EOX.3418. The opinions and assertions contained here are the private ones of the author and are not to be construed as official or reflecting the views of the Navy Department or the U.S. Government. Address correspondence to Samuel A. Lewis, Research Publications Branch, NAMRU 3 United States Naval Medical Research Unit, PSC 452, FPO, AE 09835, USA. Internet:
[email protected] Received 5 July 1995; accepted 14 March 1996.
Figure. 1 Coomassie blue-stained polyacrylamide gel. Molecular weight standards (Bio-Rad) were run in each lane. Lanes marked M were rinsed for 1 min with methanol while the other lanes were kept in destain. Gel was photographed through a yellow filter (Wratten No. 15). 820 BioTechniques
Samuel A. Lewis United States Naval Medical Research Unit 3 Code 101F FPO, AE, USA
Modified Crush-and-Soak Method for Recovering Oligodeoxynucleotides from Polyacrylamide Gel BioTechniques 21:820-822 (November 1996)
Oligodeoxynucleotides are often purified by polyacrylamide gel electrophoresis (PAGE). The two commonly used methods for recovering oligodeoxynucleotides (ODN) from polyacrylamide gels are the electroelution and the crush-and-soak methods (1). Electroelution is fast but inconvenient, as it requires a special apparatus. The crush-and-soak method is simple but time-consuming. In a standard crushand-soak method, a gel fragment containing the ODN is excised, crushed and soaked overnight in sodium acetate buffer to extract the oligodeoxynucleotide. Here, we describe a modified crush-and-soak method that eliminates the overnight soaking yet gives comparable yields. For our method, the ODN-containing gel fragment is swollen at 90°C for 5 min, frozen at -70°C for 5 min, and ODN-recovered by centrifugation. We tested this approach for the purification of a 22-nucleotide ODN and obtained yields comparable to those using an overnight soak. The ODN used in this comparison had previously been purified by PAGE so that there would be no truncated ODN, unincorporated nucleotides and cleaved protecting groups that might interfere with the spectrophotometric analysis of the ODN stock solution. Eight aliquots each containing 10 µL of 33 µM ODN and 10 µL of 0.01% bromophenol blue in 95% formamide were loaded and run into a 20% denaturing polyacrylamide gel. Following electrophoresis, the ODN was identified by ultraviolet (UV) shadowing with a handheld short-wavelength UV lamp. The ODN-containing bands could be easily seen under UV light with the gel still attaching to the lower glass gel plate (regular window glass) against a black background. The target ODN band was excised with a sharp scalpel and placed in a column filter (Quick-Snap Column; IsoLab, Akron, OH, USA). The gel slices Vol. 21, No. 5 (1996)
Table 1. Comparison of the Recovery of ODN from Polyacrylamide Gel Using the Modified Method and the Standard Method
Modified Method Loaded (nmol) Recovered (nmol) Recovery (%)a
0.33 0.278 ± 0.014 84.2 ± 4.2
Standard Method 0.33 0.287 ± 0.009 87.0 ± 2.8
aThe
difference in ODN recoveries is not significant by Student’s t-test (P >0.05, n = 4).
Table 2. Procedures for Recovering Oligonucleotides from Polyacrylamide Gel Using the Modified Crush-and-Soak Method
(1) Separate the oligodeoxynucleotide by PAGE. (2) Locate the ODN by UV shadowing. (3) Excise gel slice containing DNA. (4) Grind the gel slice in a disposable column filter. (5) Add 0.5 mL of 0.1 M Na acetate (pH 6.0). (6) Incubate at 90°C for 5 min and then at -70°C for 5 min. (7) Thaw and spin at 3000× g for 5 min to collect ODN-containing filtrate.
(approximately 10 × 4 × 0.75 mm3) were crushed with a glass rod against the side wall of the column and ground as fine as possible. The crushed gel was suspended in 0.5 mL 0.1 M Na acetate (pH 6.0). Four of the samples were processed by the modified method as follows: the columns were incubated at 90°C for 5 min in a water bath with the cap perforated to prevent pressure
Figure 1. UV scan of the oligonucleotide-containing filtrates obtained by the modified and the standard crush-and-soak methods. Solid lines indicate scans of the filtrates prepared using the standard procedure and dashed lines indicate scans of the filtrates prepared using the modified method. A control filtrate containing no ODN was subtracted from all scans to produce the above traces. Vol. 21, No. 5 (1996)
build-up inside the column. The columns were removed from the water bath, vortex mixed and then transferred to -70°C for 5 min. Subsequently, the samples were rapidly thawed at 90°C and centrifuged at 3000× g for 5 min to recover the oligonucleotide-containing filtrate. The DNA-containing filtrate could be used directly in various applications or concentrated as needed. The remaining four columns were worked up using the standard crush-and-soak procedure. To accomplish this, the columns were shaken (225 rpm) at 37°C for 15 h, and the filtrate was recovered by centrifugation. The yield of ODN was determined spectrophotometrically (ε260 nm = 4.63 µM/optical density [OD] unit). Figure 1 shows the UV scans of the eight filtrates after baseline subtraction of a control filtrate prepared from a gel strip containing no oligonucleotide. As shown, all eight recovered samples are of similar purity with an absorbance maximum at 260 nm. Table 1 shows the yields of the ODN recovered by the modified and the standard overnight soaking methods. The modified method had a recovery of 84%, while the overnight soaking method had a recovery of 87% (P >0.05). These results indicate that the modified method produces
Benchmarks similar yield as the standard method. Therefore, using this modified method, we can reduce the soaking time from 15 h to about 10 min. In summary, a simple modification of the crush-and-soak method allows the recovery of ODN from polyacrylamide gel within 30 min with high yield. The key difference in our procedure is the replacement of the overnight soak with a heat/freeze step (summarized in Table 2). We assume that heating causes the crushed gel to swell rapidly, hastening the diffusion of the ODN. Subsequent freezing may shrink the solid gel matrix while expanding the water volume, thus squeezing the ODN out of the gel network. This modified method is simple and fast, yet produces similar recovery as the standard overnight soaking method. It provides a rapid alternative to the standard crushand-soak method for recovering either labeled or unlabeled oligonucleotides from polyacrylamide gel. REFERENCE 1.Sambrook, J., E.F. Fritsch and T. Maniatis. 1989. Recovery of synthetic oligonucleotides by electrophoresis through a denaturing polyacrylamide gel, p. 11.23-11.2. Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
This work was supported by NIH Grant 1R29AI34278-01A2 (D.E.R.) and a Research Fellowship from the University of Utah (Z.C.). Address correspondence to Duane E. Ruffner, University of Utah, Department of Pharmaceutics and Pharmaceutical Chemistry, 421 Wakara Way, Suite 318, Salt Lake City, UT 84108, USA. Received 4 December 1995; accepted 1 March 1996.
Use of Engineered Thrombin Cleavage Site for Determination of Translational Reading Frames BioTechniques 21:822-824 (November 1996)
We describe a general method for the empirical determination of the translational reading frame within any cloned gene. Verification of the reading frame, particularly in the carboxy-terminal coding region, is especially important in situations where there is conflicting DNA sequence information (for example, from discrepancies that may have resulted from GC compressions). The utility of the method was demonstrated using the glpR gene of E. coli (GenBank® Accession No. M96795, P09392 or A30282). An oligonucleotide linker encoding a thrombin cleavage site and hexahistidine tag was fused in the predicted reading frame to the Cterminal coding region of the gene. Cleavage at a restriction site just preceding the thrombin site with SalI or AccI (both enzymes recognize the same sequence but leave 4-nucleotide (nt) or 2-nt overhangs, respectively), followed by fill-in and ligation reactions, yields +1 or +2 frameshifts. Susceptibility of each GlpR variant to cleavage by thrombin was then used to determine which reading frame encoded the thrombin site, thereby defining the reading frame of glpR. We wanted to verify the reading frame in the C-terminal coding region of glpR because the sequence predicted a molecular weight of 28 046, while the apparent size determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was 30 000
(3). Also, the nucleotide sequence has predicted a C-terminal cysteine residue, which is uncommon. There are several potential stop codons in other reading frames downstream of the putative stop codon of glpR. Furthermore, there are discrepancies among the sequences submitted by different research groups to the GenBank. The sequences reported may contain reading frameshifts in the C-terminal coding region due to the presence of several GC compression regions that are difficult to resolve on sequencing gels. Therefore, an approach was devised for verification of the reading frame at the C-terminal coding region of glpR. Primers TTTAcAtATGAAACAAACACAACGTCAC (5′ primer with an NdeI site) and GCAGGAgtcGacCAGCTCCAGTTGAATATGG (3′ primer with an SalI site replacing the predicted stop codon; mismatched bases in lower case) were used to amplify glpR. The PCR product was cloned into the expression vector pT7-7 (4) between NdeI and SalI, yielding pGZ115. An oligonucleotide pair encoding a thrombin cleavage site and a hexahistidine tag (Figure 1) was ligated between the SalI and HindIII sites of the above plasmid, resulting in pGZ117. pGZ117 was then digested with SalI or AccI, which recognized the same site. The resulting 4-nt or 2-nt overhangs were filled in followed by blunt-end ligation reactions. As a result, the plus-one and plus-two frameshift variants were created immediately upstream of the oligonucleotide pair, resulting in plasmids pGZ118 and pGZ119. The above manipulations were predicted to create new PvuI or NruI restriction sites in pGZ118 and pGZ119, respectively. This was verified by digestion with the appropriate enzymes. The translational reading frame of the glpR gene in the
Zhidong Chen and Duane E. Ruffner University of Utah Salt Lake City, UT, USA
Figure 1. Oligonucleotide pair encoding a thrombin cleavage site. After insertion into a plasmid, cleavage with SalI or AccI, followed by filling in and ligation reactions, generates +1 or +2 frame shift, respectively, upstream of the thrombin site. 822 BioTechniques
Vol. 21, No. 5 (1996)
