This video will describe a practical and novel method to extract genomic DNA from new BDP 100 plasma protein preservation system referred to as P 100 tubes.TNA. Extraction from serum more plasma tubes has been a pretty common procedure in the past, but the combination of this new P 100 tube and the extraction method presented here offers a novel, easier approach to retrieving better quality DNA with much less effort. The inside of the P 100 tube is coated with the potassium EDTA and protease inhibitors.
This prevents coagulation and stabilizes plasma proteins. Blood is collected in these tubes for the purpose of retrieving in testing plasma after centrifugation. Blood contained in a P 100 will separate into layers based on density of part plasma at the top, red blood cells on the bottom and white blood cells.
The Buffy coat in between these two layers. Retrieval of plasma is generally the primary use of these tubes. Several measurements are made using this plasma, such as antibody and protein levels.
The buffy coat is generally discarded, but as will be shown in this video, contains enough DNA for several analyses including sequencing, genotyping, biomarker identification, and so on. P one hundreds are comprised of three main Components, cap tube and separator. The mechanical separator migrates to just above the buffy Coat layer.
To obtain access to the buffy coat, it is necessary to increase the distance from the separator to the buffy coat. This is accomplished with the addition of 17%sucrose. The buffy coat is normally discarded with the P 102, but as will be shown in this video, it is possible to easily retrieve enough DNA from this layer to perform several Analytical tests prior to retrieving P 100 tubes from the minus 80 freezer.
Preheat a water bath to 37 degrees Celsius place each P 100 tube into the hot water bath and incubate for around five minutes to thaw all samples. Once all samples are completely thawed, any serum remaining above the separator needs to be removed. Next five milliliters of 17%sucrose is added above The separator after adding sucrose, Centrifuge, all tubes for 20 Minutes at 2, 500 G.Now that the separator Is at the top of the tube and freed from the buffy coat, it can be removed easily.
It spray 70%ethanol on a chem wipe and sterilize a paperclip hook by thoroughly wiping the entire surface. Hold the P 100 tube at a 45 degree angle with the sterile paperclip. Begin working the separator out of the tube by gently pulling up on the lip of the separator and guiding it into a biohazard bag.
With the separator out, remove as much of the 17%sucroses layer as possible without disturbing the buffy coat or red blood cell layers, the buffy coat should now be the top most layer in the tube. Transfer this layer to a new labeled 15 milliliter conical vial. The volume of buffy coat removed for each tube will vary, but should be less than three milliliters.
To normalize the volume of all tubes, add enough PBS phosphate buffered saline to bring the volume of each tube to three milliliters. Now that all buffy coats are suspended in an equal volume, any red blood cells remaining must be removed by addition of nine milliliters. Red blood cell RBC lysis solution.
Next, invert tubes several times throughout a 10 minute room temperature incubation to separate the lysed red blood cells from Buffy code Centrifuge for five minutes at 2, 500 G centrifuge in creates a pellet from which DNA will be extracted. Pour this waste out of each tube and into a liquid biohazard receptacle, leaving a small volume into which the pellet will be resuspended. Vortex the tube to dislodge and resuspend the pellet.
Add three milliliters cell lysis solution and vortex vigorously for at least 10 seconds. Having lysed the cells proteins and DNA are now freely suspended within solution and must be separated, begin by precipitating proteins using one milliliter protein precipitation solution, and VORTEXING oh high for at least 15 seconds. Now, centrifuge the tubes for five minutes of 2, 500 G to pellet the proteins during this centrifuge.
Step appropriately. Label one 15 milliliter conical vial for each sample being processed into each new tube. Transfer three milliliters, 100%isopropanol.
Once the proteins have been precipitated and centrifuged, each tube should have a relatively large pellet and a transparent mostly watt supernatant decant, the DNA containing supernatant for each tube into the corresponding isopropanol tube prepared during the last spin, the isopropanol should cause DNA in the solution to precipitate a small white wispy strings invert all tubes 50 times to ensure good mixing next centrifuge, all tubes for three minutes at 2, 500 G to create a DNA pellet, the DNA is usually visible as a small white pellet on the bottom of the tube. However, a low DNA yield may be invisible to the eye. Continue with all extractions until concentrations are measured.
Regardless of the presence of A DNA pellet, discard the isopropanol s natin. Watch the DNA by adding 70%ethanol. This will remove any remaining unwanted components that may interfere with later analysis centrifuge for one minute at 2, 500 G.To ensure the DNA pellet is attached well to the bottom of the tube, discard all 70%ethanol from tubes, then invert on a clean paper towel to air dry for one hour.
There should be no visible ethanol remaining in the tubes when continuing the protocol. The final step in the extraction is resus suspending the DNA in a solution. Either DNA hydration solution or water are adequate options for most extractions.
Add 250 microliters DNA hydration solution less for samples, which produce little or no visible DNA wisps or pellets gently and incrementally vortex the DNA to suspend. Finally, transfer all suspended DNA to a fuge tube. At this point, the concentration and purity of DNA may be read by NanoDrop or other spectrophotometer.
Generally, purity of DNA is expected to be close to that of the DNA extracted directly from whole blood obtained in potassium EDTA tubes collected for the sole purpose of retrieving DNA concentration depends on the amount of starting material, but can range from 50 nanograms per microliter to nearly 1000 nanograms per microliter averaging in the 200 nanograms per microliter range. The benefit of using this protocol is twofold. First, it allows clinicians to use a single blood draw for two clinical tests instead of drawing blood twice.
Secondly, it simplifies the process of obtaining access to DNA containing cells by using modern mechanical separator tubes rather than previous generations. Gel separator tubes with the previous generation of tubes, both the gel separator itself and a blood clot acted as barriers to the DNA containing cells. With P 100 tubes, the mechanical separator is easily removed using density separation with 17%Sucrose.
This is a screenshot Of the nano drop reading of an average DNA extraction. Using this method per sample P 109, the extraction resulted in a 250 microliter solution of 209 nanograms per microliter concentration of DNA. The total yield was around 52 micrograms.
The two 60 over two 80 ratio, the primary measure for DNA purity fell within the optimal range of 1.8 to 1.9 at 1.88. A secondary measure for DNA purity. The two 60 over two 30 ratio was near the expected range of two to 2.2 at 2.35.
These are fairly typical results. Realistically, extractions will vary in yield in quality, depending on the volume And state of blood prior to extraction using standard gel electrophoresis With the thio bromide staining. Further quality testing shows that genomic DNA extracted from P 100 tubes is as intact as DNA extracted from whole blood.
This is evident from the bright thick band appearing at the top of each lane containing genomic DNA. There are two major benefits of using this protocol in place of previous methods. First, it combines what would typically be two blood draws into one.
Secondly, this protocol is faster and cleaner than a protocol involving typical SST gel separator tubes By removing the necessity of breaking up c clotted blood routinely, clinicians draw two tubes of blood when genetics and serological tests are required. Generally, an SST gel separator tube called a gold top and a potassium EDTA tube called a purple top syrup is removed from the SST tube and DNA extracted from the purple top tube. Using this method is necessary only to draw a single red top P 100 tube.
While institutions familiar with the gel separator method of DNA extraction may be already drawing one tube for both serological and genetic tests. The method presented here is an incremental but substantial improvement over these existing gel separator methods. Both methods require removal of some kind of separator, though gel tends to be messier than plastic gel.
Separator tubes require clotted blood be homogenized through a wire mesh prior to DNA extraction. This homogenization can be messy, and DNA extraction proves more difficult when red blood cells, which contain no DNA are present in a notable quantity. This P 100 extraction method allows separation of DNA containing white blood cells from hematocrit, leaving few red blood cells to interfere with DNA extraction.
This system of retrieving DNA from a P 100 plasma tube instead of previous generation gel separator tubes has many advantages. P 100 tubes contain EDTA, which inhibit magnesium dependent endonuclease and anticoagulant to prevent blood clotting. The end result is higher DNA integrity in a much easier extraction protocol, namely the avoidance of busting up and homogenizing clotted blood.
Additionally, the plasma depleted blood may be stored in P one hundreds, indefinitely tested to three years until extraction of DNA is desired. Furthermore, clinicians would generate more useful patient data with fewer blood draws and therefore less stress to the patients undergoing the Procedures.
