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Identification of yeast DNA stand initiation site

Posted by star on 2018-09-07 01:13:53
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    In 1980, Ronald W. Davis and coworkers provided significant new insights into eukaryotic DNA replication when they discovered yeast DNA sequences, called autonomously replicating sequence elements(ARS elements), that allow plasmids containing selectable genes to replicate autonomously in yeast cells. The experiment illustrates one method for demonstrating the existence of ARS elements. Two sets of plasmids, one containing an ARS element and LEU and the other just containing LEU, are introduced into leu- yeast cells, which are then spread on agar growth medium lacking leucine. A large number of colonies form when cells transformed with ARS-plasmids are spread on the agar medium because the plasmids can replicate autonomously. In contrast, very few colonies appear when plasmids lacking ARS are spread on the agar medium and the colonies that do form contain cells in which LEU is integrated into the yeast genome.

    Yeast cells have approximately 400 ARS elements, while cells from higher animals probably have thousands. Most yeast ARS elements are named by using a number that designates their chromosome and position on the chromosome. Although the ARS elements were initially named because of their ability to support autonomous plasmid replication in yeast, they function as replicators, tors in yeast and overlap sites of replication initiation. There are two main reasons why each yeast chromosome requires several ARS elements while the E. coli chromosome requires just one replicator. First, yeast forks migrate about 30 times more slowly than those in E. coli. Second, yeast chromosomes are much longer than the E. coli chromosome.

    ARS plasmids that lack centromeres are preferentially retained by the mother cell, leading to low copy numbers in the daughter cells and high copy numbers in the mother cells. When ARS plasmids also contain centromeres, more equal plasmid segregation is observed between moth......

Two-dimensional gel electrophoresis can locate origins

Posted by star on 2018-09-06 23:37:50
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In 1987, Bonita J. Brewer and Walton L. Fangman devised a technique known as neutral/neutral two-dimensional gel electrophoresis that locates origins of replication in chromosomes, This technique was used to show that ARSs act as initiation sites for DNA replication in living cells. DNA isolated from unsynchronized rapidly proliferating cells is digested with a restriction endonuclease. Only a small fraction of any given fragment population will be derived from DNA caught in the act of replication and will contain replication forks. These replicating fragments, which vary in size from n to 2n, are first separated according to size by low-voltage agarose gel electrophoresis at neutral pH. Then the lane containing the separated fragments is cut from the gel, rotated 90°, and placed at the top of a new gel for separation in a second dimension.

The new gel contains a higher agarose concentration and is run at high voltage at neutral pH, allowing it to separate fragments by shape, Therefore, fragments that migrated together in the first dimension separate from one another in the second with those containing bubbles migrating at the slowest rate, those with a single replication fork(Y-shaped) at an intermediate rate, and those that are linear duplexes at the fastest rate. Once migration is complete, the fragments are transferred to a positively charged nylon membrane and the migration of a specific fragment is detected by hybridization with a DNA probe that is labeled with a radioactive or fluorescent tag.

Two types of replication intermediates are readily distinguished from the observed patterns. When a replication origin is located within the boundaries of the restriction fragment, the bubble-shaped replication intermediates produce a bubble arc pattern. When a replication fork is passively generated from an origin of replication that is outside the fragment, the Y-shaped replication intermediates generate a Y-arc (probe 2).

Practice for Exhaust Recycling

Posted by star on 2018-09-06 23:33:52
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1.Perform agarose gel/ethidium bromide electrophoresis to fractionate DNA fragments. Any type or grade of agarose may be used. However, it is strongly recommended that fresh TAE buffer or TBE buffer be used as running buffer. Do not reuse running buffer as its pH will increase and reduce yields.

2. When adequate separation of bands has occurred, carefully excise the DNA fragment of interest using a wide, clean, sharp scalpel. Minimize the size of the gel slice by removing extra agarose.

3. Determine the appropriate volume of the gel slice by weighing it in a clean 2mL

microcentrifuge tube. Add 1 volume Binding Buffer(XP2). Incubate at 55-65℃ for 7 minutes or until the gel has completely melted Vortex or shake the tube every 2-3 minutes.

4. Insert a HiBind DNA Mini Column in a 2 ml Collection Tube.

5. Add no more than 700μL DNA/agarose solution from Step 5 to the HiBind DNA Mini Column.Centrifuge at 13,000 x g for 1 minute at room temperature

6. Discard the filtrate and reuse collection tube.

7. Repeat Steps 7-9 until all of the sample has been transferred to the column.

8. Add 300 μL Binding Buffer (XP2). Centrifuge at maximum speed(13,000 x g) for 1 minute at room temperature. Discard the filtrate and reuse collection tube.

Note: SPW Wash Buffer must be diluted with 100% ethanol prior to use.

9. Centrifuge at maximum speed for 1 minute at room temperature. Discard the filtrate and reuse collection tube.

10. Centrifuge the empty HiBind DNA Mini Column for 2 minutes at maximum speed to

dry the column matrix

Note: It is important to dry the HiBind DNA Mini Column matrix before elution.

Residual ethanol may interfere with downstream applications.

11. Transfer the HiBind DNA Mini Column to a clean 1.5 mL microcentrifuge tube.Add 15-30 μL Elution Buffer or deionized water directly to the center of the column membrane.

12. L......

Research Found Key Substances in the Pathogenesis of Psoriasis

Posted by star on 2018-09-06 18:53:36
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    A new study in Japan found that in the pathogenesis of psoriasis, a signaling molecule in skin cells plays a key role, and on this basis, it is expected to develop new therapeutic methods.

    Psoriasis is a skin disease associated with immune abnormalities, manifested by erythema, silver shavings and itching on the skin. According to the researchers, psoriasis patients account for about 3% of the global population. In recent years, injection antibody therapy has a good effect, but the cost of treatment is high, and some patients may have antibody failure.

    In a new issue of Epithelial TRAF6 drives IL-17–mediated psoriatic inflammation on the Journal of JCI Insight, the researchers reported that a signal molecule "TRAF6" in skin cells plays a key role in the pathogenesis of psoriasis.

    In mice, mice lacking this signaling molecule do not develop psoriasis. Even when these mice were injected with immunomodulatory factors, immune abnormalities that could have caused psoriasis were induced, and psoriasis was still suppressed.

    Researchers therefore believe that new treatments for psoriasis can be developed for this signaling molecule to address the challenges of previous related therapies.

    Wuhan EIAab Science Co., Ltd has developed several related kits such as E8869h, E8869m, E0063h and E2103h etc. Welcome scientific research workers to choose and purchase.




    Recently, the Chinese research team found that phage lyase can kill bacteria that cause dental caries, but it is harmless to common oral symbiotic bacteria, and it can be confirmed by rat dental caries model to significantly reduce the degree of dental caries.

    Caries, commonly known as cavities or fangs, are oral diseases in which teeth are broken and gradually form cavities. They are the main cause of tooth loss. Oral diseases, including caries, have a great impact on our quality of life. According to the researchers, the production of dental caries is mainly caused by the long-term interaction of bacteria with food. The main caries are Streptococcus mutans and Streptococcus sobrinus.

    This study was based on the previous discovery that phage lyase has a broad spectrum of Streptococcus mutans, and further tested the killing effect on other oral fungi and common oral commensal bacteria. The results showed that phage lyase deformed common caries Streptococcus mutans and Streptococcus sobrinus and related biofilms have bactericidal effects, but have almost no killing effect on oral symbiotic bacteria such as Streptococcus sanguinis, Streptococcus oralis, and Streptococcus salivarius.

    The study found that phage lyase also kills Enterococcus faecalis that causes periapical periodontal infection and eliminates biofilm. Related research Activity of the Chimeric Lysin ClyR against Common Gram-Positive Oral Microbes and Its Anticaries Efficacy in Rat Models were recently published on Virus. This research means that phage lyase has a good prospect in the prevention and treatment of dental diseases such as dental caries, and it may provide new drugs and tooth care products for the p......

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