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31.
Identification and characterisation of PmaCI an endonuclease of novel specificity from Pseudomonas maltophila. 总被引:3,自引:3,他引:0
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We report the use of MonoQ FPLC (Fast Protein Liquid Chromatography) for the rapid purification of a novel Type II restriction endonuclease PmaCI, from Pseudomonas maltophila, which recognises the sequence 5'-CAC decreases GTG-3'. The resulting enzyme is free of other nucleases to a level suitable for its characterisation by multiple-substrate digestion and DNA sequencing techniques. This method appears to be widely applicable and we have used it for the isolation of restriction endonucleases of comparable purity from a range of other organisms. Also described is a rapid method for screening a library of small inserted regions in recombinant M13 molecules for the presence and subsequent screening of restriction sites of interest. 相似文献
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A Bacillus subtilis plasmid that can be packaged as single-stranded DNA in Escherichia coli: use for oligodeoxynucleotide-directed mutagenesis 总被引:1,自引:0,他引:1
A Bacillus subtilis/Escherichia coli shuttle vector was modified to contain the origin of DNA replication of the E. coli filamentous phage f1, in both orientations. Upon superinfection with and f1-related phage of an E. coli strain containing either of the modified vectors, the single-stranded (ss) form of the plasmid was packaged in virions and released to the culture medium. Each of these ss DNAs has been purified from the virions and used as a template for oligodeoxynucleotide-directed mutagenesis. The resulting mutations were demonstrated by DNA sequencing. The capacity of these vectors to be isolated as phage ss DNA from E. coli and to replicate as plasmids in B. subtilis makes them convenient substrates for the production of oligodeoxynucleotide-directed mutations for studies in B. subtilis. 相似文献
33.
Efficient extraction of RNA from mammalian tissue 总被引:10,自引:0,他引:10
Marsha L. Frazier Wendy Mars Dagne L. Florine Richard A. Montagna Grady F. Saunders 《Molecular and cellular biochemistry》1983,56(2):113-122
RNA extraction from mammalian tissue has been compared using the different deproteinizing agents: a) guanidine-HCl, b) guanidinium-thiocyanate, c) buffer-saturated phenol, or d) buffer-saturated phenol followed by a proteinase K digestion of the aqueous phase. Both solid tissues (first, second, and third trimester fetal bovine pancreas), and human white blood cell populations were studied. Degradation, as seen in citric acid-urea agarose gels, and the ability to serve as templates for cell-free protein synthesis were used as criteria to assess the efficiency of the different methods. We conclude that employing buffer-saturated phenol with proteinase K digestion is a superior method for consistent extraction of relatively undegraded RNA in quantitative amounts from mammalian tissue. 相似文献
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The identification and quantitative analysis of abscisic acid in plant extracts by gas-liquid chromatography 总被引:1,自引:1,他引:0
Summary New techniques are described which permit the quantitative analysis of microgram quantities of abscisic acid in plant extracts by gas chromatography. Presumptive methyl abscisate peaks on gas chromatograms are positively identified by photosensitised isomerisation to methyl 2-trans-abscisate. Losses of abscisic acid during pre-purification are corrected by using 2-trans-abscisic acid as an internal standard. 相似文献
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In a variety of tumour systems, individuals carrying progressively growing neoplasms have lymphoid cells with a specific cytotoxic effect on cultured tumour cells from the same individual1–4. Since the sera of tumour-bearing individuals have been shown to prevent tumour cell destruction by immune lymphocytes in vitro2,5–8 and since this serum blocking activity appears early in primary and transplant tumour development5,7, it has been suggested that the appearance of this serum blocking activity might be responsible for the progressive growth of tumours in individuals having cytotoxic lymphocytes. Counteraction of this blocking activity would thus be of primary importance in facilitating the function of an already existing or bolstered cell-mediated immunity. The serum blocking activity might be inhibited in various ways, by preventing the formation of blocking antibody or by interfering with its action (“unblocking”), as demonstrated in Moloney sarcoma regressor sera9. This type of serum also has a therapeutic effect on Moloney sarcomas in vivo10,11, which has been tentatively attributed to its unblocking activity8,9 or, possibly, to a complement-dependent cytotoxicity10. Tumour growth in the Moloney sarcoma system, however, might be due in part to continuous recruitment of neoplastic cells by virus-induced transformation and so the therapeutic effect could be due to a virus-neutralizing serum activity9,10. 相似文献
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