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Source CiteSeerX
Content type Text
File Format PDF
Subject Domain (in DDC) Computer science, information & general works ♦ Data processing & computer science
Subject Keyword Ols Pool ♦ Construction Primer ♦ Assembly Pcr ♦ Incorrect Product ♦ 5-20 Ng ♦ 20-30 Cycle ♦ Subpool Amplification ♦ Total Chip-eluted Dna ♦ Dna Concentration ♦ Secondary Band ♦ Full-length Product ♦ Different Synthesis Product ♦ Important Factor ♦ Robust Assembly ♦ 2-3 Order ♦ 15-20 Thermal Cycle ♦ Ols Material ♦ Preassembly Step ♦ Pre-assembly Step
Abstract We found that three factors were critical for the robust assembly of OLS Pool 2 subpools into full-length products (data not shown). First, using a preassembly step of 15-20 thermal cycles performed in the absence of construction primers followed by 20-30 cycles of assembly PCR with the construction primer reduced formation of incorrect products. Second, using low annealing temperatures (50-55 °C) during the pre-assembly and more stringent annealing temperatures during the assembly PCR (60-72 °C) also helped reduce the formation of secondary bands. Third, using an increased DNA concentration (5-20 ng/!L) in the pre-assembly step (2-3 orders of magnitude greater than for OLS Pool 1) was the most important factor in improving the reliability between different synthesis products. Differences in quantity of OLS material utilized between OLS Pools 1 and 2. While the 13,000 features in OLS Pool 1 can be used to construct>700 genes, each subpool amplification used 1/500 th of the total chip-eluted DNA.
Educational Role Student ♦ Teacher
Age Range above 22 year
Educational Use Research
Education Level UG and PG ♦ Career/Technical Study