Cloning protocol

As host cells that have been successfully transformed proliferate, they rapidly amplify the recombinant DNA. For example, under favorable conditions of nutrient availability and temperature, a single recombinant plasmid introduced into an E. coli cell can be replicated a billion times in about 11 hours. However, transformed and untransformed cells usually have identical appearances. (See Figure 18C for an example of an exception to this mle.) Consequently, researchers often design cloning protocols utilizing vectors with selectable marker... 

PCR Cloning Protocols From Molecular Cloning to Genetic Engineering, edited by Bruce A. White, 1996... 

RT-PCR Protocols, edited by Joe O Connell, 2002 192. PCR Cloning Protocols, 2nd ed., edited by Bing-Yuan Chen and Harry W. Janes, 2002... 

Cherbas and Cherbas (1998) provides a brief account of the histories of the cell lines with speculations on the histological origin(s) of the embryonic lines and with a tabular account of much that is known about gene expression in various lines. For a broader biological review of the lines, see Schneider and Blumenthal (1978), Sang (1981), Cherbas and Cherbas (1981), and Echalier (1997). For cell maintenance and cloning protocols, see Cherbas and Cherbas (1998), and for transformation protocols, see Cherbas et al. (1994), supplemented, for the S2 expression system, by Kirkpatrick and Shatzman (1999). 

Ausubel, F. M., Brent, R., Kingston, R. E., etal., eds., 1987. Current Protocols in Molecular Biology. New York John Wiley Sons. A popular cloning manual. 

Figure 3.3 Comparison of array CGH among DNA extracted from fresh tissue, FFPE tissue by heating protocol or nonheating protocol for two human tissue samples of metastatic carcinoma in lymph node (a-c), and undifferentiated non-small cell carcinoma (d-f). Array CGH hybridization genomic profiles show ratio values representing relative copy number of single BACs. A good result is scored as 1.0 that indicates a low standard deviation for gains (>0.2), normal (0.0), or losses (<-0.2). In these two cases, fresh samples show best score as 2, both FFPE tissue samples show identical score of 3. Each spot represents the average of three replicates. Clones are ordered by chromosomal position as numbers at the bottom (x axis) of each picture. The y axis is the log2 ratio of test reference intensity. Provided by Sandy DeVries from Dr. Frederic Waldman s Lab at UCSF.

Sambrook, J. and Russell, D. 2006. Condensed Protocols from Molecular Cloning A Laboratory Manual. Cold Spring Harbor Laboratory Press, USA. 

Cloning of constructs was performed according to standard protocols [Sambrook et al. 1989], The mutations were created in the plasmid pBluescript II KS (+) by oligonucleotide-directed mutagenesis. 

In the following, a short overview of the expression cloning method with Xenopus laevis oocytes is given. Protocols for standard procedures are provided in the Appendix, while those methods that are subject to frequent modification are described in general and the reader is referred to the corresponding handbooks for further details. 

In vitro transcription from cloned cDNA sequences is required to produce mRNA that can be injected into Xenopus oocytes for expression of the encoded protein and is achieved by standard procedures. While oocyte isolation and injection generally follows the protocols provided below, in vitro transcription into mRNA may be modified in accordance to the instructions provided with... 

Figure 24.1 Flow chart of RACE-PCR protocol adopted with slight modifications from the instruction manual of the Marathon cDNA amplification kit. Poly A+ RNA is used for the generation of an adaptor-ligated cDNA library. Fragments containing the 5 - and the 3 -end of the cDNA coding for the carrier are amplified from the uncloned library with adaptor primers and gene-specific primers. Afterwards, a full-length clone is generated from the individual RACE products by subcloning or end-to-end PCR.

The enzyme catalyzing the formation of retinal 2 by means of central cleavage of P-carotene 1 has been known to exist in many tissues for quite some time. Only recently, however, the active protein was identified in chicken intestinal mucosa (3) following an improvement of a novel isolation and purification protocol and was cloned in Escherichia coli and BHK cells (4,5). Iron was identified as the only metal ion associated with the (overexpressed) protein in a 1 1 stoichiometry and since a chromophore is absent in the protein heme coordination and/or iron complexation by tyrosine can be excluded. The structure of the catalytic center remains to be elucidated by X-ray crystallography but from the information available it was predicted that the active site contains a mononuclear iron complex presumably consisting of histidine residues. This suggestion has been confirmed by... 

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