Lpcrrflp Performance and Analysis

Amplifying large DNA fragments requires high-fidelity DNA polymerase enzymes, e.g., TAKARA LA (Takara). The conditions for LPCR amplification require optimizing primer, magnesium ion, and enzyme concentrations as well as thermal cycling number and timing. In a typical LPCR reaction protocol, 1-100 ng of template DNA is amplified in the presence of 1 mM of each primer, 300 mM of dNTPs, 2.5 mM MgCl2, and 1.5 U of TAKARA LA polymerase. The amplified LPCR products are purified on agarose gels, then are digested with a restriction enzyme, e.g., Clal. The digested DNA fragments are then electro-phoresed on 6% polyacrylamide gel for a constant period of time at constant voltage. A reference DNA marker is run along with the digested DNAs in the first, middle, and last lanes to help identify the size of the restricted DNA fragments. After staining with ethidium bromide, the restriction pattern is visualized and photographed with a digital camera.

Standardization of electrophoretic conditions, i.e., using the same lot of precast polyacrylamide gel, and applying the same voltage, e.g., 50 V for the same period of time, e.g., 6.5 hr for each run, is very important to obtain reproducible banding patterns. The positions for all DNA fragments in each restriction pattern are determined and digitized by fingerprinting software, e.g., Dendron (Solltech). The program assigns a molecular weight value for each DNA fragment in each restriction pattern using the reference DNA marker. A similarity coefficient (SAB)

Table 1 LPCR primers and their 5' and 3' coordinates in relation to Variola genome (GenBank LL22579)

Segment

Forward primer

Reverse primer

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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