Dna isolation protocol from virus

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A convenient tool to build experimental workflows and find products to match your needs. Log Out. Show More. Log in to see your account pricing. Kit Accessories. Carrier RNA. Spin column procedure. Vacuum procedure. These products are not intended for the diagnosis, prevention, or treatment of a disease. High sensitivity in PCR. Generally, the transfer vector method is preferable when only a small number of genetic modifications are required. In this study a rapid and reliable method was developed to extract baculovirus DNA for PCR analysis from cultured insect cells.

The method was tested for direct screening of recombinant viruses in plaque assays. Contrary to previous reports, baculovirus DNA was isolated directly from viral plaques and successfully analysed by PCR.

After the ultracentrifugation, you should see a thin opaque pellet with a dark spot in the middle. Carefully aspirate the liquid from the tube, including drippings along the sides, but avoiding the pellet at the bottom of the tube. Let the pellet sit for at least 10 minutes to allow hydration of the pellet. Break up the pellet by pipetting with a small-bore pipette tip e. Do not worry about shearing at this step, because the viral DNA is still contained in capsids at this point.

Add the following reagents to the tube containing the viral pellet, at room temperature, to destroy capsids:. Beware of shearing from here on, e. Extract viral proteins by adding 5 ml of phenol-chloroform and immediately begin inverting or vortexing to maintain an emulsion. Mix for 10 seconds, then centrifuge the emulsion for 5 minutes at rcf at room temperature.

Collect the top layer with a wide-bore pipette tip, avoiding the interface, and transfer to a new tube. Optional: Use phase lock gel PLG tubes in this step to avoid contamination from the interface. Add 10 ml of ice-cold ethanol, cover the tube e. Watch for a DNA "ghost" to appear, which consists of a thin ropy precipitate. Invert the tube gently to mix further and cause the sticky DNA precipitates to cluster together.

Use a glass hook to capture the DNA ghost s. Carefully blot the DNA to remove excess liquid, then place the hook tip down into a 1.

Add 0. Allow resuspension of DNA to proceed for at least one hour. Optional: To maximize DNA yield, break off the glass hook into the 1. Once the infected cell pellet is harvested, the remaining protocol steps can be complete in one long day or carried out over two days Figure 2. When preparing glass hooks to capture the DNA ghost, it is important to make the hook angle be less than 90 degrees, so that later it can fit into the base of an 1.

Sealing the tip of this hook prevents DNA loss inside the pipet. DNA ghosts form during the precipitation step and will appear as white, ropy threads Figure 4. Thus the same glass hook can be used to gather up multiple threads of DNA from within the precipitation solution. Table 2. Preparation of TNE 0. Table 3. Preparation of PBS 2. Figure 1. Figure 3. Three representative examples of glass hooks used to collect the viral DNA ghosts. Figure 4. Representative example of a well-formed and abundant DNA "ghost".

Similar extractions are commonly used for RNA viruses as well. The robustness of this DNA preparation likely results from the multiple methods of purification that are included. The initial Freon extractions denature lipid membranes, separating cell components and also releasing the lipid envelope and outer tegument proteins that surround herpesvirus capsids. This is followed by ultracentrifugation, where the heavy viral nucleocapsids pellet through two gradient cushions, effectively separating them from most other cellular components.

These capsids are resuspended, and the viral nucleocapsid DNA is released by rupturing the capsids with proteinase K and detergent. Two phenol-chloroform extractions thoroughly remove nucleocapsid components and any remaining cellular proteins. Finally, precipitation of the large viral DNA genomes in solution reduces carryover of salts or particulate contaminants.

This procedure to isolate viral nucleocapsid DNA provides abundant, high purity material for downstream applications. The combination of multiple extraction and centrifugation steps distinguishes this protocol from simpler single-extraction protocols that leave more cellular debris or degraded protein products with the DNA Protein contaminants can decrease the storage stability of viral DNA, and reduce transfection efficiency into cells.

The quantity of cells used for infection impacts the eventual yield of viral DNA. For viral mutants with a decreased yield of infectious virions, the input material should be scaled upward accordingly. Doubling the input number of dishes will approximately double the yield, and can be processed with the same quantities of reagents described here. To increase the input beyond this point, we recommend separating the input material into two parallel handling streams e.