This video demonstrates a procedure to isolate high purity viral nucleo capsid, DNA from infected cells. First lipid membranes are denatured using a dual free on extraction. The sample is then ultracentrifuge to pellet the viral nucleo capsids and separate them from lighter cellular and viral components.
Detergents and proteinase K are added to release the viral DNA from the pelleted nucleo capsids. Then dual phenyl chloroform extractions are performed to remove all remaining proteins. Finally, the viral DNA is precipitated in solution.
Ultimately, the results show the highly concentrated and pure viral DNA has been isolated from these infected cells. The main advantage of this technique over existing methods that do not involve free un extractions or ultracentrifugation, is that the viral DNA produced here has very low levels of host DNA contamination, making it ideal for downstream procedures such as PCR and deep sequencing of viral genomes. We first used this method in the early days of recombinant DNA work when extra precautionary measures were of high importance.
We developed this procedure to isolate viral DNA while working in a glove box under BSL four conditions. Visual demonstration of this method is critical because the gradient formation and DNA ghosting steps are difficult to learn. This is because layering the gradient requires an extremely steady hand and the DNA ghost that precipitates in solution can be hard to see if you don't have prior experience spotting it.
Begin this procedure by preparing five to 10 15 centimeter diameter dishes of tissue culture cells for infection. Here, PK 15 cells are prepared for infection with pseudo rabies virus when the cells are 95 to 100%confluent, replace the medium with four milliliters of medium containing virus. At an MOI of five to 10, incubate the plates for one hour in a humidified 37 degrees Celsius incubator at 5%CO2 every 15 minutes.
Gently rock the plates to ensure that the cell mono layer remains fully coated by the virus inoculum. During the incubation, warm the medium for the next step. After one hour of infection, aspirate the viral inoculum from the plates.
Then add 15 milliliters of warm medium and incubate at 37 degrees Celsius for 12 to 20 hours in a humidified incubator. Finally, in preparation for the next day's experiments, prepare a fresh batch of LCM and TNE buffers. Note that addition of NP 40 will result in the formation of ribbons in the acquis solution.
Place the prepared solutions on a rocker at four degrees Celsius overnight. By the next day, the solutions should be fully integrated The next day. Place the dish on the stage of a compound microscope and visually confirm that infection of cells has caused uniform cytopathic effect.
All of the cells should be rounded up, but should not have lifted off the plate. If the cells have not yet rounded up, continue the incubation until cytopathic effects are observed. Next, scrape the infected cells into the medium and combine cells in medium into 50 milliliter conical tubes to increase the yield.
Rinse the plates with 10 milliliters PBS and combine with the cells in medium, then centrifuge cells and medium for 10 minutes at 2000 RCF at room temperature to rinse away any remaining medium and serum aspirate, supernatant and resuspend the pellet in 10 milliliters of PBS. Repeat centrifugation and aspiration. If the experiment will not be continued on the same day, store the pellet at minus 20 degrees Celsius until it is used for the steps shown in this section.
It is important to keep the cell pellet and LCM buffers on ice whenever possible. Prepare for ultra ification by adding Beamer capto ethanol to 0%glycerol LCM buffer so that the final concentration of beam or capto ethanol is 0.043%Also prepare the ultracentrifuge by setting it to four degrees Celsius and turning on the vacuum while the ultracentrifuge is cooling. Add five milliliters of 0%glycerol LCM buffer with 0.043%Beamer capto ethanol to the cell pellet and pipette up and down to resuspend the pellet.
Be sure to break up all of the clumps if necessary. Vortex the tube to break up any undissolved cell pellet. Next to extract the cellular and viral membranes, add 1.5 milliliters.
Freon a vortex vigorously immediately. Centrifuge for 10 minutes of 2000 RCF at four degrees Celsius. Following the spin, there will be two layers.
The top acquiesce layer contains viral nucleo, capsis and proteins, while the bottom Freon layer contains viral and cellular membranes. Using a wide bore pipette tip, collect the top layer avoiding the interface and transfer to a fresh 50 milliliter conical tube. It is important to avoid touching the Freon layer and carrying over that organic solvent to the acquiesce layer in the new tube.
Add freon and vortex as before to repeat the Freon extraction following centrifugation. Collect the top layer approximately five milliliters and transfer to a fresh poly aamer ultracentrifuge tube. Add beamr capto ethanol to 0.043%to the two remaining glycerol LCM buffers.
Next, to prepare a gradient in the polymer centrifuge tube, use a thin pipette to underlay the Freon extract with three milliliters of 5%glycerol LCM. Then underlay this with 2.5 milliliters of 45%glycerol LCM. Also, if needed, prepare an equivalent balance tube using the same proportions of LCM buffers.
Place the tubes into ultracentrifuge buckets and balance them to within 0.1 grams of each other by gently adding extra 0%glycerol LCM buffer to the top layer 50 to 100 microliters at a time. Spin the balanced samples for one hour at 77, 000 RCF at four degrees Celsius during ultracentrifugation. Use a glass past pipette to make a hook to capture floating DNA at the ethanol precipitation.Step.
Hold both ends of the pipette and suspend the middle section over flame. When a section of glass is warm, pull gently to stretch the glass. Bend it gently to create a tightly angled hook of less than 90 degrees and pull sharply to seal off the end.
If the tip of the glass hook is open, hold the tip over the flames so that the glass melts enough to close it. After the ultra ification, there will be a thin, opaque pellet with a dark spot in the middle, which contains the viral nucleo. Capsids carefully aspirate the liquid from the tube, including drippings along the sides, but avoiding the pellet at the bottom of the tube.
Add 0.5 milliliters TNE at room temperature. Let the pellet sit for at least 10 minutes to allow hydration of the pellet. If the procedure will not be continued until the next day, place the resuspended pellet at four degrees Celsius.
Using a P 200 tip, break up the hydrated pellet by pipetting up and down. Do not worry about shearing at this step because the viral DNA is still containing capsids at this point. To break open the capsids, add the following to the viral pellet, 4.25 milliliters, TNE 0.25 milliliters 10%SDS and two milligrams of protein EK at room temperature.
Gently rock. To mix these reagents. There should be a slight increase in viscosity now that the capsids have been destroyed, care should be taken to avoid sharing the DNA use wide boar pipettes and do not vortex the viral material to extract the viral proteins.
Transfer the sample to a phase lock gel tube and add five milliliters of phenol chloroform. Immediately begin inverting to maintain an emulsion mix via inverting or for at least 10 seconds. Then centrifuge the emulsion for five minutes at 2000 RCF at room temperature.
Following the spin, take the top layer and transfer it to a new phase lock gel tube. Repeat the phenol chloroform extraction and centrifuge again after the second phenol chloroform extraction. Collect the top layer into a 30 milliliter glass coex tube and chill the tube at minus 20 degrees Celsius for 10 to 15 minutes.
Taking care that it does not freeze. Add 10 milliliters device cold ethanol para. Film the tube and invert to mix.
Immediately watch for a DNA ghost to appear, which consists of a thin ropey precipitate. Invert the tube gently to mix further and cause the sticky DNA precipitates to cluster together. Finally, use a glass hook to fish out the DNA ghosts carefully blot the DNA by dabbing it with a kim wipe to remove excess liquid.
Then place the hook tip down into a 1.5 milliliter tube. To maximize DNA yield, break off the glass hook into the 1.5 milliliter tube so that resus suspension off the glass Fragments can continue over time. Allow to dry add 0.25 to 0.5 milliliters of te to dissolve the DNA ghost.
Allow resuspension of DNA to proceed for at least one hour store DNA at four degrees Celsius. Viral DNA was prepared from nucleo capsids as described in this video article shown here is a representative example of a well-formed and abundant DNA ghost consisting of linear viral genomic DNA precipitated in solution. The strands cluster together and will require time to rehydrate once placed in an Acquia solution prepared viral DNA from PRV strains, Becker Bartha, and four additional strains was run for one hour on a 0.5%agros gel and stained with athenia bromide.
Each PRV sample uses one microliter of a viral DNA nucleocapsid preparation 250 microliters total volume on the left. A ladder of lambda bacteria phage. DNA sty one digested demonstrates the large size of undigested lambda DNA 48.5 KILOBASE pairs and PRV genomic DNA approximately 140 kilobase pairs.
Next, a dilution series of known quantities of Lambda DNA demonstrates that the concentration of the PRV samples on the right varies from 50 to 200 nanograms per microliter for a total yield of 12 to 50 micrograms per viral. DNA preparation prepared viral DNA samples from four PRV strains were digested overnight with either BAM H one or KPN one. Restriction enzymes digested DNA samples were separated by overnight electrophoresis on a 0.8%agros gel and stained with athe and bromide bam.
H one lanes include eight micrograms of viral nucleocapsid, DNA, while the KPN one lanes include four micrograms viral DNA Each different PRV strains can be distinguished by their respective restriction fragment length polymorphism patterns. Following this procedure, other methods like deep sequencing can be performed in order to obtain data on viral genome content After its development. This technique paved the way for researchers in the field of virology to characterize viral DNA by restriction, fragment length polymorphisms or R fps, southern blotting, cloning recombinant, DNA virus construction, and other manipulation of herpes viruses and other types of viruses.
Don't forget that working with fon can be extremely hazardous and precautions such as working in a fume hood should always be taken while performing this procedure. After pelleting, the fon layer can quickly change from a gelatinous semi-solid to a liquid, so each tube should be evaluated individually before deciding whether pouring or pipetting is the best route to collect the aqueous upper layer.
