PCR reaction

Figure 4.7. In vivo recombinational cloning in yeast. Two successive PCR reactions are performed. Each set of primers contains 5 flanking sequences that are eventually used for homologous recombination with vector sequences after transformation of yeast with the 2nd PCR fragment and the linearized vector.

Carter, D.A., Burt, A. and Taylor, J.W. (1995) Direct analysis of specific bands from arbitrarily primed PCR reactions. In Innis, M.A., Gelfand, D.H. and Sninsky, J.J. (eds) PCR Strategies. Academic Press, New York, pp. 325-332. 

Prepare 25 fil PCR reactions using 5 fil diluted PAT assay as template. Use standard PCR components and buffers supplied with the Fast-start Taq (Roche, Basel, Switzerland). Cycling conditions are 2 min at 95° then 30 s each at 95,60, and 72° for 25 to 35 cycles (template-dependent) 2 min 72°. 

Multiplex PCR a PCR reaction where more than one primer set is included in the reaction pool allowing 2 or more different DNA targets to be amplified by PCR in a single reaction tube. 

PCGC-2 software package, 7 448 PC resins, properties of, 10 196t PCR reaction, 12 514 PC-SAFT equation of state, 24 11 PCT Gazette, 18 236 PCT molding resins, 20 60-61. See also Poly(cyclohexanedimethylene-terephthalate) (PCT)... 

Exogenous sources such as a person s hair or skin, doorknobs, laboratory benches, dust, reagents, thermal cyclers, and pipet tips are some of the common sources of DNA contamination. Ideally, a laminar air flow bench with filtered air provides a clean, dust-free environment. Sample preparation should be done in a separate room or area. The addition of sample to the PCR reaction mixture in the... 

Another possibility to enhance the specific DNA-fragment is to perform a nested PCR. With this PCR method, sequences of very low abundancy can be amplified, but the risk to enhance the background at the same time is very high. For a nested PCR, a small aliquot of the first PCR reaction is used together with gene-specific primers which bind within the initial PCR product. 

PCR reaction using natural dNTPs. However, there are some drawbacks to epPCR. Generally, the technique produces libraries of DNA fragments which need to be ligated into expression plasmids (this can be a limiting step), and some of the methods do not produce random mutations. 

Examples of proprietary enzymes include Mutazyme II DNA Polymerase (www. stratagene.com). Other companies have developed kits containing selections of reagents which can be combined to yield controlled error rates in PCR reactions. 

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