Cloning efficiency

The cloning efficiency (CE) of the control culture is calculated as follows ... 

On day 8, each culture is again trypsinized, counted and diluted so that a sample cell population can be assessed for cloning efficiency and a second sample can be assessed for the induction of 6TG-resistant cells. 

For estimation of cloning efficiency and mutant induction, cells are plated out in 96-well microtiter plates. Flasks and microtitre plates are incubated at 37°C in a C02 incubator as in the cytotoxicity assays. 

The calculation for cloning efficiency is made as for the cytotoxicity assay. 

Relative total growth (RTG) is a cytotoxicity parameter which considers growth in suspension during the expression time and the cloning efficiency of the end of the expression time as follows ... 

Not unexpectedly, addition of large quantities of recombinant SCF to TFl cocultures on parental MMCE synthesizing no SCF or transduced SI SCF did not increase the cloning efficiencies of TFl cells. Addition of recombinant SCF, even in excess of 100 ng/ml to cocultures of TFl on MMCE cells transduced to present mb SCE could not impair the growth promoting potential of these feeder cells (Eig. 2d). 

To investigate the cloning efficiency of the progenitor cells in these cocultures we analyzed their colony formation ability in methylcellulose supplemented with recombinant growth factors. The results indicate that within the first two weeks mb SCF as well as SI SCF or S1+MS5 stroma induced... 

Figure 6. Proliferation of the murine Myl-D7 stem cell line is stimulated efficiendy only by membrane-bound CSF-1. 2-10 Myl-D7 cells were cultured on MMCE cells expressing the different CSF-1 isoforms in 48-well plates. Cloning efficiencies were determined weekly and Myl-D7 clones (>10 cells) were transferred onto new MMCE cells. Values of Myl-D7/MS5 cocultures were set to 100% at each time point (data not shown). Results are expresses as percentage of control Myl-D7/MS5 cultures and are mean values +SD (four experiments). After 3 weeks in coculture a) no difference was found in the proliferation rates of cells stimulated by either wildtype- or soluble CSF-1 (P>.05) b) mean values for populations stimulated by membrane-bound- or soluble CSF-1 were significantly different (P<.001).

The assay principle should, however, be applicable to any target hapten, unlike assays based on a chemical modification. Cloning efficiency of the hybridoma-secreting anti-idiotype antibodies would be in a practical range, and much higher than that of anti-metatype antibodies. We (Kl) established four kinds of a-type and two kinds of /3-type anti-idiotype antibodies after three fusion experiments, each using spleen cells from one immunized mouse. Barnard et al. (Bl, B3, B4)... 

The cloning efficiency of the present method is sufficient to obtain positive clones from a single culture set, although artificial alterations of nucleotide sequences originating from the PCR process are inevitable. 

We recommend introducing the Kpnl recognition sequence into the C-terminal primer if the target cDNA does not have a Kpnl site, because this strategy has been shown to have the highest cloning efficiency. 

For certain aspects of work with cell cultures, a useful property of tunicamycin is that it can be used to synchronize cell division. After release of the block imposed hv the drug, the cloning efficiency was also higher, and the cloning size more regular, than in the control culture.547... 

Tates, A.D., van Dam, F.J., de Zwart, F.A., van Teylingen, C.M.M. Natarajan, A.T. (1994) Development of a cloning assay with high cloning efficiency to detect induction of 6-thio-guanine-resistant lymphocytes in spleen of adult mice following in vivo inhalation exposure to 1,3-butadiene. Mutat. Res., 309, 299-306... 

We have found that cloning efficiency is reduced when increased volumes of purified PCR product are used, possibly as a result of carryover of contaminants from the cleanup reaction. Therefore, it is best to have the PCR product as concentrated as possible. The exact amount of DNA in this step can vary somewhat to allow for standardized pipeting volumes without compromising cloning efficiency. 

The exact amount of DNA in this step can vary somewhat to allow for standardized pipeting volumes without compromising cloning efficiency. 

Lipids also can be beneficial for cells in culture, since some substances absorbed by the cells need to be solubilized in lipids, or in some cases the toxicity of compounds may be reduced by complexation with lipids. The absence of essential lipids such as linoleic acid, lecithin, cholesterol, ethanolamine, or phosphorylcholine can result in the decrease of cloning efficiency and in reduction in the size of colonies, as shown for insect cells by Echalier (1997). However, one of the difficulties in supplying lipids at reasonable concentrations is their low solubility. To circumvent this limitation, lipids can be emulsified with complexing agents such as Pluronic F68 or cyclodextrin (Maiorella et al., 1998). 

The introduction of DNA fragments into an appropriate vector and the E. coli transformation (Figure 17.7) require basic molecular biology techniques [68]. However, particular attention must be paid to maximizing the cloning efficiency, especially for the construction of large libraries. 

Increasing the concentration of NaCl in the medium led to a steady decrease in cloning efficiency. Likewise, the two compatible solutes glycerol and inositol led to decreased cloning efficiency as their concentrations were elevated. Urea and glycine betaine showed complex indi-... 

Liang H et al (1996) Wavelength dependence of cell cloning efficiency after optical trapping. Biophys J 70(3) 1529-1533... 

Cloning efficiency The percentage of cells plated (seeded, inoculated) that form a clone. One must be certain that the colonies formed arose from single cells in order to use this term properly. See Colony forming efficiency . 

Plating efficiency This is a term that originally encompassed the terms Attachment (Seeding) efficiency. Cloning efficiency and Colony forming efficiency... 

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