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In vitro translation more efficient with additional tRNA
technical tips
TIG - - September 1986 ,
Isolation of yeast DNA
In vitrotranslation more efficient with additionaltRNA Rabbit reticulocyte lysate is a well known system for translating,/m ~,o, .m._~Asfrom a rode variety of cells. Endogenous tRNAin the reticulocytelysate is u~uallysuf~cientto ensure some translation. However, it can be important for some purposes to obtain the ~ u m possble translational activity, for exampie in monitoring the purification of mRNA or characterizing the products of recombinant DNA clones. It was mentioned briefly in the ofiginalpaper of H. R. B. Pelham and IL J. Jackson [Eur. ]. B~kem. 67, 247 (1976)] that the addition of extra tRNA can improvethe results, and we have noticed that addition of crude yeast tRNA (Sigma) approximately doubles the efficiencyof translation of yeast mRNA. This is particularly obvious for high molecular weight proteins (see Fig. I). The degree to which added tlLNA enhances translational activityappears to depend on the source of the in vib'o translation kit used, perhaps indicatingthat some suppliers' kits already contain extra tRNA.
1
2
3 4
,|
5 6
Fig. ,. AutoradiogrOhofSDSPAGF, ofm vitrotr~l~ion~oduds ~lled wi~ l~S]metk~ine, sln~i~ tke effect of" adding yeast ffNA (lpg, I0 r~ mU) to tramla~ommi,~ures contoiningl pgSaccharomycescerevisiae po~A) r~NA. Lane 1, no RNA; lane 2, control from yeast v~NA providedin NEN kit; lone3, Contributedby M. Crouzet and N. S. cerevisiae~/y(A)mRNA; lane4, Bataille, Laboratoirede C~n~tique, poly(A)mRNA ~th yeast~NA; lane Universit~ de Bordeaux !1, Allee 5, onlyyemt ~RNAadded;iane6, for des Facult~s,33405TalcnceCedex, comparison, protein labelledin vivo France.
K, phenol-¢Moroformextraction and a final ethanol precipitation. Proteinase K treatment and the volume of the phenol extraction are important factors for obtaining the maximum yield. The DNA obtained cuts well with restriction endonucleases. It is claimedthat the method is rapid, can be scaled up for one-litre 'ogphase cultures, and is applicable to other cells with tough cell walls, such as the green alga Cklmydommms and the fungus Neuros~ora crassa.
This method, which is adapted from the procedure of Kielland. Brant et al. [(1979) Carlsberg Res. Commun. 44, 77-87], yieldsboth plasrnidand highmol. ecular weight chromosomal DNA from Sacclmromyces ceve. ~/ae and is claimed to give 759b of the theoretical yield. The method involves treatment of yeast cells with zymolyase to produce spheroplasts, iysis of the spheroplasts in the presence of the chaotropic agent guani. diniumhydrochlorideto denature protein and inhibit nucleases, Holm, C., Meeks-Wagner, D. W., precipitation with ethanol, re- Fangman, W. L. and Botstein, D. moval of protein with proteinase (1986)Geme42, 169-173
Sectioningyolk-ladenovaries,eggsand embryosfor insituhybndization The yolk-ladenoocytes and embryos of various animals (e.g. Dros~kila. Xevml~s) are ~,fficult to section when standard parat~ embedding is used. Two salient features seriously hampersatisfactory sectioning:, the density of protein-rich yolk bodies, and the lack of connective structures within the nutritive stores. Thus in most publishedrnicrographsof hybridization on such sections, the cytological details appear ~orly preserved. These hindrances may be removed by the following proceduret. (1) Selected ovaries, eggs or embryos are fixed in a freshly prepared 49~ formaldehydesohtion: paraformaldehyde (analy. tical grade) is depolymerized in double-distilled water at 70°C'-, the solutionneutralized and mixed with 0.~-M S~rensen's phosphate buffer pH 7.0 (Dros~ki/a) or pH 7.2 (X'enopm). (2) The washed specimens are carefully embedded in a thick agar gel prepared from a filtered solutionat 60°C (1.5 g of purified agar in 100 ml of distilledwater). (3) The t~nmed agar blocks are gradually dehydrated with
50% ethanol, 709~ethanol, then 2-ethoxy-ethanoL (4) After optimalimmersionin butan-l-ol, they are embedded in a mixture of synthetic stearates, Steedman's 'Ester wax 196(W3. Infiltration is carded out under increasingvacuumas the solvent is far less volatile than toluene. (5) The trimmed blocks are sectioned in the same way as paraffin blocks. The serial sections are flattened on slides and dried, and the wax is then removed in toluene baths before hydration. Clean gelatinized slides are used for routine work; polylysine gives a lower hackground than gelatin on autoradiographs, but is more expensive.
References I P~tavy,G. (1985)StainTeck~l. 60, 39-1-330 2 Pease, D. C. (1962)Anat. Rec. 142, 342 3 Steedman, H. F. (1960)Se~m Cuing in Microscopy,BlackweH
Contributed by Georges P~tavy, ER 229, CNRS, F-91190, Gifsur-Yvette, France.
A sensitive non-radioactive method for detecting DNA* Biotin-labelled DNA probes on nylon or nitrocellulose filters can be detected by BhGENE®. Such probes are stable for at least one year, unlike most radioactive probes. Bethesda Research Laboratories claim that the high-activity strep. tavidin-a~,~line phospbatase con228
jugate on which BhGENE® is based allows detection d less than 0.25 pg DNA, and is more
sensitive than other non.radio. active methods. Bethesda Research Laboratories, LifeTechnotogiesInc., POBox6009 Gaithersburg, MD 20877, USA. *Basedoninformationsuppliedbythe manufacturer.
TIG - - September 1986 ,
Isolation of yeast DNA
In vitrotranslation more efficient with additionaltRNA Rabbit reticulocyte lysate is a well known system for translating,/m ~,o, .m._~Asfrom a rode variety of cells. Endogenous tRNAin the reticulocytelysate is u~uallysuf~cientto ensure some translation. However, it can be important for some purposes to obtain the ~ u m possble translational activity, for exampie in monitoring the purification of mRNA or characterizing the products of recombinant DNA clones. It was mentioned briefly in the ofiginalpaper of H. R. B. Pelham and IL J. Jackson [Eur. ]. B~kem. 67, 247 (1976)] that the addition of extra tRNA can improvethe results, and we have noticed that addition of crude yeast tRNA (Sigma) approximately doubles the efficiencyof translation of yeast mRNA. This is particularly obvious for high molecular weight proteins (see Fig. I). The degree to which added tlLNA enhances translational activityappears to depend on the source of the in vib'o translation kit used, perhaps indicatingthat some suppliers' kits already contain extra tRNA.
1
2
3 4
,|
5 6
Fig. ,. AutoradiogrOhofSDSPAGF, ofm vitrotr~l~ion~oduds ~lled wi~ l~S]metk~ine, sln~i~ tke effect of" adding yeast ffNA (lpg, I0 r~ mU) to tramla~ommi,~ures contoiningl pgSaccharomycescerevisiae po~A) r~NA. Lane 1, no RNA; lane 2, control from yeast v~NA providedin NEN kit; lone3, Contributedby M. Crouzet and N. S. cerevisiae~/y(A)mRNA; lane4, Bataille, Laboratoirede C~n~tique, poly(A)mRNA ~th yeast~NA; lane Universit~ de Bordeaux !1, Allee 5, onlyyemt ~RNAadded;iane6, for des Facult~s,33405TalcnceCedex, comparison, protein labelledin vivo France.
K, phenol-¢Moroformextraction and a final ethanol precipitation. Proteinase K treatment and the volume of the phenol extraction are important factors for obtaining the maximum yield. The DNA obtained cuts well with restriction endonucleases. It is claimedthat the method is rapid, can be scaled up for one-litre 'ogphase cultures, and is applicable to other cells with tough cell walls, such as the green alga Cklmydommms and the fungus Neuros~ora crassa.
This method, which is adapted from the procedure of Kielland. Brant et al. [(1979) Carlsberg Res. Commun. 44, 77-87], yieldsboth plasrnidand highmol. ecular weight chromosomal DNA from Sacclmromyces ceve. ~/ae and is claimed to give 759b of the theoretical yield. The method involves treatment of yeast cells with zymolyase to produce spheroplasts, iysis of the spheroplasts in the presence of the chaotropic agent guani. diniumhydrochlorideto denature protein and inhibit nucleases, Holm, C., Meeks-Wagner, D. W., precipitation with ethanol, re- Fangman, W. L. and Botstein, D. moval of protein with proteinase (1986)Geme42, 169-173
Sectioningyolk-ladenovaries,eggsand embryosfor insituhybndization The yolk-ladenoocytes and embryos of various animals (e.g. Dros~kila. Xevml~s) are ~,fficult to section when standard parat~ embedding is used. Two salient features seriously hampersatisfactory sectioning:, the density of protein-rich yolk bodies, and the lack of connective structures within the nutritive stores. Thus in most publishedrnicrographsof hybridization on such sections, the cytological details appear ~orly preserved. These hindrances may be removed by the following proceduret. (1) Selected ovaries, eggs or embryos are fixed in a freshly prepared 49~ formaldehydesohtion: paraformaldehyde (analy. tical grade) is depolymerized in double-distilled water at 70°C'-, the solutionneutralized and mixed with 0.~-M S~rensen's phosphate buffer pH 7.0 (Dros~ki/a) or pH 7.2 (X'enopm). (2) The washed specimens are carefully embedded in a thick agar gel prepared from a filtered solutionat 60°C (1.5 g of purified agar in 100 ml of distilledwater). (3) The t~nmed agar blocks are gradually dehydrated with
50% ethanol, 709~ethanol, then 2-ethoxy-ethanoL (4) After optimalimmersionin butan-l-ol, they are embedded in a mixture of synthetic stearates, Steedman's 'Ester wax 196(W3. Infiltration is carded out under increasingvacuumas the solvent is far less volatile than toluene. (5) The trimmed blocks are sectioned in the same way as paraffin blocks. The serial sections are flattened on slides and dried, and the wax is then removed in toluene baths before hydration. Clean gelatinized slides are used for routine work; polylysine gives a lower hackground than gelatin on autoradiographs, but is more expensive.
References I P~tavy,G. (1985)StainTeck~l. 60, 39-1-330 2 Pease, D. C. (1962)Anat. Rec. 142, 342 3 Steedman, H. F. (1960)Se~m Cuing in Microscopy,BlackweH
Contributed by Georges P~tavy, ER 229, CNRS, F-91190, Gifsur-Yvette, France.
A sensitive non-radioactive method for detecting DNA* Biotin-labelled DNA probes on nylon or nitrocellulose filters can be detected by BhGENE®. Such probes are stable for at least one year, unlike most radioactive probes. Bethesda Research Laboratories claim that the high-activity strep. tavidin-a~,~line phospbatase con228
jugate on which BhGENE® is based allows detection d less than 0.25 pg DNA, and is more
sensitive than other non.radio. active methods. Bethesda Research Laboratories, LifeTechnotogiesInc., POBox6009 Gaithersburg, MD 20877, USA. *Basedoninformationsuppliedbythe manufacturer.