| pubmed-article:365684 | rdf:type | pubmed:Citation | lld:pubmed |
| pubmed-article:365684 | lifeskim:mentions | umls-concept:C0014834 | lld:lifeskim |
| pubmed-article:365684 | lifeskim:mentions | umls-concept:C0032136 | lld:lifeskim |
| pubmed-article:365684 | lifeskim:mentions | umls-concept:C0080194 | lld:lifeskim |
| pubmed-article:365684 | lifeskim:mentions | umls-concept:C1510411 | lld:lifeskim |
| pubmed-article:365684 | lifeskim:mentions | umls-concept:C0012854 | lld:lifeskim |
| pubmed-article:365684 | lifeskim:mentions | umls-concept:C1521761 | lld:lifeskim |
| pubmed-article:365684 | lifeskim:mentions | umls-concept:C0392760 | lld:lifeskim |
| pubmed-article:365684 | pubmed:issue | 4 | lld:pubmed |
| pubmed-article:365684 | pubmed:dateCreated | 1979-3-13 | lld:pubmed |
| pubmed-article:365684 | pubmed:abstractText | The susceptibility of E. coli strain chi1776 to transformation by pBR322 plasmid DNA was examined and optimized. Maximum transformation to tetracycline (Tc) resistance was achieved when cells were harvested from L broth at 5.0--6.0 . 10(7) cfu/ml, followed by washing twice in cold 0.1 M NaCl + 5 mM MgCl2 + 5 mM Tris, pH 7.6. Cells grown in the presence of D-cycloserine (Cyc) rather than nalidixic acid (Nx) transformed markedly better. The presence of 5 mM Mg2+ ions in washing and CaCl2 solutions stimulated transformation about 2-fold. Optimal conditions for transformation included a pH range of 7.25-7.75 and a cell-to-DNA ratio of about 1.6 . 10(8) cfu/ng plasmid DNA. The frequency of transformation was highest when cells were exposed to 100 mM CaCl2 in 250 mM KCl + 5 mM MgCl2 + 5 mM Tris, pH 7.6, before mixing with DNA. A 60 min incubation period for cell + DNA mixtures held on ice produced the maximum number of Tcr transformants. In our hands, heat shocks at 37 degrees C or 42 degrees C for various times all decreased transformation to about one-half of optimal levels. Furthermore, the recovery of transformants was best when cell + DNA mixtures were plated on precooled (4 degrees C) Tc agar plates. The efficiency of plating was optimum when only 5 microliter of cell + DNA mixture was spread per plate, suggesting that non-viable background chi1776 cells on selective medium inhibited the recovery of transformants. It was also found that the presence of linear DNA molecules in cell + DNA mixtures markedly inhibited the transformation of chi1776 by pBR322 plasmid DNA. On the basis of these findings, a new procedure for the plasmid-specific transformation of E. coli chi1776 by pBR322 plasmid DNA is proposed. The use of this technique has allowed us to attain transformation frequencies in excess of 10(7) transformants/microgram pBR322 plasmid DNA. | lld:pubmed |
| pubmed-article:365684 | pubmed:language | eng | lld:pubmed |
| pubmed-article:365684 | pubmed:journal | http://linkedlifedata.com/r... | lld:pubmed |
| pubmed-article:365684 | pubmed:citationSubset | IM | lld:pubmed |
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| pubmed-article:365684 | pubmed:status | MEDLINE | lld:pubmed |
| pubmed-article:365684 | pubmed:month | Jul | lld:pubmed |
| pubmed-article:365684 | pubmed:issn | 0378-1119 | lld:pubmed |
| pubmed-article:365684 | pubmed:author | pubmed-author:MonahanJ JJJ | lld:pubmed |
| pubmed-article:365684 | pubmed:author | pubmed-author:KeenJJ | lld:pubmed |
| pubmed-article:365684 | pubmed:author | pubmed-author:NorgardM VMV | lld:pubmed |
| pubmed-article:365684 | pubmed:issnType | Print | lld:pubmed |
| pubmed-article:365684 | pubmed:volume | 3 | lld:pubmed |
| pubmed-article:365684 | pubmed:owner | NLM | lld:pubmed |
| pubmed-article:365684 | pubmed:authorsComplete | Y | lld:pubmed |
| pubmed-article:365684 | pubmed:pagination | 279-92 | lld:pubmed |
| pubmed-article:365684 | pubmed:dateRevised | 2006-4-13 | lld:pubmed |
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| pubmed-article:365684 | pubmed:year | 1978 | lld:pubmed |
| pubmed-article:365684 | pubmed:articleTitle | Factors affecting the transformation of Escherichia coli strain chi1776 by pBR322 plasmid DNA. | lld:pubmed |
| pubmed-article:365684 | pubmed:publicationType | Journal Article | lld:pubmed |
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