Key

| Deletion " Primers

80% efficiency mutated plasmid isolation Fig. 4 Inverse PCR mutagenesis 2. Schematic for the deletion of a region within a plasmid.

would involve introducing random nicks that can be caused by heating[1] or limited DNase treatment. However, a major drawback to this approach is the difficulty in controlling the degree of nicking to the construct.

The PCR component of the IPCR reaction needs to be optimized both in relation to the quantity of template, primers, and Taq polymerase, and in terms of the cycling conditions. As discussed, titration of the starting material in the PCR reactions will help to overcome some of these problems, and it is essential that appropriate controls are included in the PCR reaction. Analysis of products by electrophoresis will also elucidate any technical difficulties. A common problem is the formation of primer dimers or multimers, as is smearing recognized after the first-round PCR, which can be attributed to nonspecific amplification that maybe caused by an excess of template or inefficient digestion and/or ligation. Increased annealing temperatures as well as decreased primer and Taq polymerase concentrations may alleviate nonspecific primer binding and also minimize the formation of primer dimers, although it is recommended that when designing primers, the appropriate software packages (e.g., Net-primer: www.premierbiosoft.com/netprimer) are used to reduce the opportunity of primer dimer/polymer formation.

Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

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