Tris-EDTA

Fig. 4.3.1 Effect of pH on the total light emission of phialidin (A), and the temperature stability profiles of phialidin (minute open circles) and aequorin (solid line) (B). In A, each buffer contained 0.1 M CaCl2 plus 0.1 M Tris, glycine or sodium acetate, the pH being adjusted with NaOH or HC1. In B, the photoprotein samples in 10 mM Tris-EDTA buffer solution, pH 8.0, were maintained at a test temperature for 10 min, and immediately cooled in an ice water bath. Then total luminescence activity was measured by injecting 1ml of 0.1 M CaCl2/Tris-HCl, pH 7.0, to 10 pd of the test solution. From Levine and Ward (1982), with permission from Elsevier.

Fig ure 3. Starch gel electrophoresis of hemoglobins. Tris-EDTA-boric acid buffer, pH 9.0. O-Dianisidine stain. 

The presence of Individual chains In a hemoglobin variant can also be demonstrated by electrophoresis at alkaline pH after the protein has been dissociated Into Its subunits through exposure to 6 M urea In the presence of 3-mercaptoethanol. The buffer is either a barbital buffer or a tris-EDTA-boric acid buffer, pH 8.0 - 8.6, and contains 6 M urea and 3-niercapto-ethanol. Dissociation of the hemoglobin Into subunits Is best accomplished In a mixture of 1 ml 10 g% Hb (or whole hemolysate), 4 ml 6 M urea barbital or tris-EDTA-boric acid buffer, and 1 to 1.5 ml 3-mercaptoethanol. After 30 minutes to 1 hour the sample Is subjected to cellulose acetate or starch gel electrophoresis. Each chain has a specific mobility and an alteration In electrophoretic mobility easily Identifies the abnormal chain. 

Three pH values (acidic, neutral, and basic AR solution), and three heating conditions (under boiling, boiling, and pressure heating) are recommended for the basic test battery. However, alternative procedures may be applied according to laboratory facilities and routine protocols as described above. Currently, citrate buffer pH 6.0, Tris-EDTA buffer pH 8-9, and certain AR solutions at lower pH, such as boric acid pH 2-3, or acidic acid buffer pH 2, as well as 0.05% citraconic anhydride pH 7.4, may be used to evaluate the optimal AR protocol. 

Tris-EDTA (TE) buffer 10 mM TRIZJMA hydrochloride, 1 mM EDTA. 

Resuspend the pellet of DNA in 25 /xL Tris-EDTA, pH 8.0 buffer. Save the plasmid prep for agarose electrophoresis (part C) and use in Experiment 15. 

As the fold of the RNA is critical, the folding step after purification of the RNA is important. The most commonly used folding protocols involve heat-cooling the RNA in a metal-free buffer such as Tris-EDTA and subsequently adding metals (Ke and Doudna, 2004) such as monovalent or divalent cations, most commonly Na/K+ and Mg2+. Slow renaturation of the folded RNA from denaturing conditions may also be effective, particularly in RNA-protein systems. Both the human 5 virus ribozyme... 

Figure I. Separation of PGM and AK electrophoresis was done for 22 hr at 6.5 V/cm at 4°C in a 1 mm, 14% starch gel prepared in JM Tris, EDTA, maleic acid, MgCl, = 7.4 tank buffer diluted 1 10. The PGM side of the gel was stained at 1-2 hours before the AK using an agar overlay technique at 37°C. The visualized bands are precipitated with fotmazan.

Sucrose solution 0.25 M Tris-EDTA Pancreatic RNAase solution... 

Double-stranded products were purified using centrifugation dialysis following the protocol described by Allard et al.31 Purified double-stranded products were subsequently dried under vacuum and resuspended in 15 /A of IX Tris/EDTA buffer (TE). To obtain single-stranded DNA, we used 2 n 1 of the purified double-stranded PCR product as the template. The concentrations of Taq DNA polymerase, reaction buffer, dNTPs, and... 

Harvest the cells by centrifugation and resuspend the cell pellet in 1.5 ml of Tris/EDTA/lithium acetate ... 

Tris-EDTA (TE) buffer 10 mMTris-HQ, 1 mMEDTA, pH 8. Paraformaldehyde (4%, w/v) Boil 100 mL of PBS containing 4 g of paraformaldehyde in a fume cupboard. Cool on ice prior to use. The final pH of this solution should be 7.2-7.4 without adjustment. Proteinase K/pepsin The source used is important The activity of these enzymes varies according to manufacturer and lot We have found the proteinase K supplied by Boehringer Mannheim (FRG) the most consistent, and the details quoted in Methods refer to that source. We find that pepsin obtained from Sigma (Poole, Dorset UK) is superior to that from Boehringer (FUG) and can be used at 0.1% (w/v) compared with 0.4% (w/v) in PBS. 

Fig. 13. Starch gel electrophoresis of hemoglobin of cord blood samples from newborns with various types of a-thalassemia. Tris-EDTA-boric acid buffer, pH 8.6. Stained with o-dianisidine. From Pootrakul et al. (P22) with permission of the authors and publisher.

Fig. 15. Horizontal starch gel electrophoresis to show the small differences in mobility between three fetal hemoglobin variants. Left to right (1) Hb-F-Malta-I heterozygote, (2) and (3) normal adults (4) Hb-Fx in a Negro newborn (5) Hb-F-Malta-II heterozygote (6), (7), and (8), Hb-A, Hb-F, and Hb-F-Malta-II fractions, respectively, isolated by column chromatography on CM-cellulose. Tris-EDTA-boric acid buffer, pH 9.0, o-dianisidine stain.

The organic solvent was removed by rotary evaporation (40-45°C, 30-60 min). The plasmid DNA solution (1.5 mg/mL in Tris/EDTA buffer) was added to the dry lipid film and the lipids solubilized by vigorous agitation (see Note 3). 

Prepare the picogreen solution as described by the provider (1/200 in tris-EDTA buffer). 

Tris-EDTA buffer 50 mM Tris-HCl, pH 7.5, containing 20 mM EDTA. 

Resuspend the pellet in each tube with 9 mL Tris-EDTA buffer using a Janke Kunkel polytron tissumizer (or a similar model) equipped with a 100 mm long x 10 mm OD shaft and combine two tubes together such that each liter of original bacterial culture is now contained in four tubes. 

Carefully pour off the supernatant, resuspend the pellet in 15 mL Tris-EDTA buffer with the tissuemizer, and centrifuge again as in step 9. Repeat this three more times (see Note 34). 

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