DNA Purification With Diatomaceous Earth, PCR Screening Of Lycopene β-Cyclase From Orange Genomic DNA Library And Analysis Of Upstream Sequence

The paper is mainly divided into three parts, part one studies for the purification of DNA;part two includes employing PCR to screen the single phage which has sequence of lycopene β- cyclase gene LCY-b2 from the orange genomic DNA library, part three is the analysis of the up-stream of LCY-bl sequence with promoter database-assistant from the internet.DNA purification is an important and regular work in molecular biology labs. The second part of this thesis researched on application of diatomaceous earth purified DNA from plasmid, genome DNA samples. A lot of influence index has been researched;Appling the purification protocol of diatomaceous earth to the genomic DNA of leaves and flesh of fruit trees, and the purified DNA quality is much higher than those un-purified.The genomic library includes most of his genes;the high sensitivity that PCR reaction makes it can orient the aimed phage and clone the position effectively. This research plans to continue screening its possible allele LCY-b2. The main result of study of the experiment is as follows: the pace is 6 cm/min to subside, 1 cm/min, and DNA of particle of 0.25 cm/min absorb ability invite respectively for 0.009 μg/mg , 0.3 μg/mg, 2.5 μg/mg. Absorbing 1 jig λ DNA, adding 20 μl diatomaceous earth suspend liquid (nearly include 10 mg diatomaceous earth particle ) is most suitable, therefore the ability of absorbing of the diatomaceous earth particle is about 0.1 μg/mg. The rate of recovery reaches 77.31%. Employ this improved method purified a low copy plasmid pLZ14 for 14953 bp , At the result, plasmid purity is up to 23.87%, 11.06 times higher than un-purify plasmid. The abstraction of the fruit tree crop genomic DNA. Employ diatomaceous earth removing many phenol and impurity in the pulp from navel orange and old leaves successfully, DNA got can be used in molecular biology experiments such as PCR, etc.directly.We design primers according to LCY-b2 sequence, employ filter paper- PCR method to screen this gene from the genomic library of navel orange.;We use the optimized screening methods to improve the probability that the goal phage appears in the course of screening , but fail to found the single phage we want. But we lay a foundation for continuing screening .Utilizing software and database like promoter 2.0, NNPP,EPD SPACE, we analyzed the up-stream sequence of the navel orange lycopene beta cyclase gene up to its TSS, found its possible location of promoter and RNA poly II binding site.