Cloning steps

Many transfections required to get a diverse library. More cloning steps. 

In some circumstances the aim of the experiment will be to clone a DNA sequence which lacks cohesive ends. Such molecules may, for example, be the cDNA products from the reverse transcription of particular mRNA species and this is currently the most widely used procedure for sequencing eukaryotic mRNAs. Under controlled conditions the reverse transcriptase from Avianmyeloblas-tosis virus (AMV) will use a mRNA template to synthesize a full-length double-stranded DNA copy (cDNA) of the RNA sequence. The products of this reaction frequently lack precisely defined ends and it is necessary to trim such frayed or uneven termini before proceeding to the cloning step. Incubation with DNA polymerase (Klenow) or T4-polymerase (Section 4.2.2.) will yield molecules with clean blunt-ended termini which can be... 

The cloning of functional genes from natural microbial consortia is dependent on the high quality of the extracted and purified environmental DNA since the enzymatic modifications required during the cloning steps are sensitive to contamination by various biotic and abiotic components that are present in environmental ecosystems. For example, extraction of DNA from soils always results in coextraction of humic substances, which interfere with restriction enzyme digestion and PCR amplification and reduce transformation efficiency and DNA hybridization specificity [19 -22], Therefore, extraction methods have been developed to remove or minimize contamination of the purified DNA by humic or other interfering substances. Several protocols for the isolation of bacterial community DNA from various environmental samples have been reported in recent years. These methods are based either on re-... 

All Southern hybridizations are based on DNA extracted from thousands of individual cells. Even if the cell lines are based on a cloning step, one must be aware that none of the cell lines is clonal in the most narrow sense Genetic variations occur very rapidly in immortalized cells, due to their inherent chromosomal instability. 

Due to methodological problems it is presently not possible to quantifying those taxa which are determined to be present in clone libraries these problems, that are associated with the extraction of nucleic acids, PCR-primer sensitivity and selectivity, cloning steps, and the dependence of PCR amplificate amount on undeterminable genomic properties, have been summarized [21]. Thus, it is also not possible to decide whether the identified clones belong to a majority or a minority population of the naturally occurring prokaryotes. 

Figure 12.2 Recombinant DNA approaches for gene multimerization (a) and cloning steps involved in rPBPs expression (b). RE represents the restriction enzyme site for compatible sticky-ends. (Adapted with permission from [76], Copyright 2011, American Chemical Society.)...

A. Preparation of Cells. In order to facilitate the uptake of plasmids into bacteria, cultures are treated to make them more permeable, or competent . Competent cells may be purchased commercially and may be preferable for critical cloning steps as they are prepared using methods that generally give better uptake than the one given here. The method provided here (modified from Sambrook et al. 1989) is cheap and simple, does not use obnoxious chemicals such as dimethylsulfoxide (DMSO), and works with a wide variety of bacterial strains. We use it fbr routine maintenance of our plasmid stocks and for general subcloning. 

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