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Introduction to Plasmid DNA Preparation

High-quality plasmid DNA is very important for many researches, which can help researchers finish some biological experiments with high efficiency, such as cell transfection, DNA vaccines, antibody preparation and other preclinical studies. Genemedi provides researchers the plasmid DNA preparation services to extract high-quality DNA from different types of raw materials, including special strains. Our company can not only provide the preparation of plasmid DNA in small quantities for laboratory, but also service for bioengineering and pharmaceutical companies about large-scale plasmid DNA preparation.

Properties

Products delivered by Gene synthesis services
Product composition 1. Plasmid: High-purity lyophilized plasmid DNA containing your intended synthetic construct
2. Gene Synthesis Report sheet: Electronic PDF file and paper version A4 paper
Form Lyophilized plasmid
Instructions for use and Storage 1. According to your experiment requirements plasmid should be dissolved in ddH2O or TE with the appropriate concentration.
2. Plasmid can be quantitatively dissolved and preserved at -20℃.

Advantages

1. Flexibility: up to gram quantity.

2. Economical: 100 μg miniprep starting from just $50.

3. Flexible: Microgram to gram production.

4. Extremely low levels of endotoxin: ≤0.05 EU

5. Fast turnaround time: Only an additional 3 days with any gene synthesis order.

6. One-stop downstream solutions: combine with gene synthesis, custom cloning, or mutagenesis.

Applications and Figures

Quality control description

1. Super coiled content(electrophoresis)

2. 1.7≤OD260/OD280≤2.0

3. Restriction analysis (optional add-on)

4. Genomic DNA and RNA residual

5. Plasmid concentration

6. Sterility testing (optional add-on)

7. Endotoxin Assay (optional add-on)

Frequently Asked Questions(FAQs)

1. What is "animal-free cultivation media"? What is the advanced animal-free prep process and when would I need it?
Answer

For our research and industrial grade plasmid prep services, we use a cultivation media without any animal-derived components, making the media "animal-free." Unlike the traditional plasmid prep that uses bovine pancreatic RNase, there is bovine-free recombinant RNase during our animal-free prep process. The advanced animal-free prep process is critical for clinical applications and animal studies to avoid safety risks.

2. Why is it important to measure the supercoiled content of a DNA plasmid prep?
Answer

The supercoiled state of a DNA plasmid corresponds to its functional or active state in an application: If the prepared DNA plasmid has higher level of the supercoil, the structure of the prepared DNA plasmid is more similar to the natural DNA, and also the plasmid has better functionality. Many studies show that high-supercoiled plasmids are key to the success of numerous in vivo and in vitro applications. Supercoiling has strong influence on the efficiency of transfection and can lead to sides effects in DNA therapy. In general, the supercoil percentage has been determined as a predictor of plasmid performance and is accepted by many regulatory entities.

3. How does endotoxin affect the results of research?
Answer

Endotoxins have lots of adverse downstream effects on your research, such as decreasing the efficiencies of transformation, causing the interference about in vitro transfection of immune cells etc. So, it is important for gene research to use plasmid DNA without endotoxin.

4. How is a small amount of plasmid DNA preparation in gene cloning prepared?
Answer

There is a simple one-step extraction method, alkali lysis method and boiling method for the common plasmid DNA extraction methods. The most commonly used alkali lysis method, the principle is: in the PH 12.0-12.6 alkaline environment, linear molecular weight bacterial chromosome DNA degeneration, and covalent closed-loop plasmid (CC) DNA is still a natural state. When PH is adjusted to neutral and high salt concentration exists, the cross-linking of chromosome DNA forms an insoluble reticular structure. Most chromosomal DNA and proteins form precipitation under the action of detergent SDS, while plasmid DNA is still soluble. By centrifugation, most of the cell fragments, chromosomal DNA, RNA and protein are removed, plasmid DNA is still in the supernatant, and plasmid DNA is recovered by ethanol precipitation. A small quantity of commercially available plasmid extraction kits can help you reclaim high-quality, high-efficiency plasmids.

5. How is the method of plasmid purification selected?
Answer

The method of plasmid purification is chosen by the molecule weight of plasmid, the species of bacterium, and also the requirements of the experiments. When the molecule weight of plasmid》15kb, the bacteria are suspended in an iso-osmotic glucose solution, lysozyme and EDTA is added to destroy cell walls and cell membranes. Then the detergents, such as SDS, are added to resolve some spheroids. When the molecule weight of plasmid<15kb, boiling and alkali cracking can be used to purify plasmid. However, it’s not recommended about boiling to purify plasmid from some E. coli strains which release large amounts of carbohydrates after treatment with denaturant, lysozyme and heating. And also, the strains containing restriction endonucleases should not be purified with boiling.

6. What are the QC ( quality control) methods for testing AAV in Genemedi?
Answer

We provide qPCR-based titer as the primary method to determine whether the packaging/purification process is successful or not. If your virus has GFP or RFP reporter, we also perform virus infectivity testing in HEK293T cell line.

7. Can I utilize different serotypes of AAV virus in the same equipment to keep the infected cells?
Answer

There is no problem with using different serotypes in the same equipment, as long as the handler takes the basic precautions to avoid cross-contamination.

8. What is the difference between brain localization of gene expression after injection of AAV or lentiviral vectors?
Answer

Lentiviral particles don't spread well after stereotaxic injection into brain because the particles are relatively large (90nm~100nm). In contrast, AAV particles spread more readily due to smaller size (20nm~100nm). As mentioned, it has been stated that some AAV serotypes spread better than others in brain, for example, AAV5 is reported to spread exceptionally well when injected into striatum. Lastly, pretreatment with mannitol to your animal about 15 min ahead of viral injection has been reported to aid in spread of viral particles in the brain.

9. Do AAVs tranduce axons passing through a region of interest?
Answer

AAVs are able to infect axon terminals and produce retrograde transport (towards the cell body). The slightly longer answer is that this process is highly biased based on the serotype you are using. There are a number of papers reporting AAV6 (and maybe AAV6.2) is the main.

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