In this video, we are going to show you some tips for ensuring successful isolation of high molecular weight DNA from blood samples using the Monarch HMW DNA Extraction Kit for Cells & Blood.
Unlike many other blood protocols, the Monarch workflow begins with an erythrocyte lysis step and we will review the details of this so that you know what to expect at each stage of the process.
We’ll begin with fresh blood samples. After adding 3 volumes of cold RBC Lysis Buffer to your sample, mix immediately by inverting several times. You’ll notice that the solution is opaque. Incubate on ice until the sample starts lysing, which will be indicated by the solution starting to become translucent. Check the sample frequently, and each time you check, invert the sample a few times to mix.
As soon as you notice that the sample starts to become translucent, incubate it for an additional 3-5 minutes on ice to make sure the erythrocyte lysis is complete. The sample is now ready for the centrifugation at 4°C.
Now let’s discuss erythrocyte lysis with frozen blood samples. Leucocytes from these sample are very fragile and tend to clump easily. If they are not handled properly, yields can be negatively impacted. Add 3 volumes of cold RBC Lysis Buffer to the frozen blood sample and shake it a few times to make sure the buffer can move freely inside the tube. Place the samples in a rack at room temperature and shake them occasionally to monitor the thawing, until the buffer begins to turn red. This usually takes around 30-90 seconds.
Once the buffer starts to turn red, place the tubes in the vertical rotating mixer at about 10 rpm to facilitate thawing and set a timer to make sure that you do not exceed 5 minutes. As soon as the samples are thawed, take them out and if necessary, invert a few more times manually to complete the thawing. Vortex the samples briefly to break up any cell aggregates that may remain and place them on ice. You are now ready for the following centrifugation step.
It’s very important to work without interruption and to keep samples on ice to minimize the stress on the leukocytes during the erythrocyte lysis.
Another important point when processing blood samples is that it is essential to remove the supernatant properly after the erythrocyte lysis in order to get optimal results. Carefully remove the supernatant while keeping the pellet facing down and keeping the pipette tip on the upper side of the tube.
Leave the last 15-20 µl in the tube to avoid removing any part of the leucocyte pellet which can be relatively loose. This is what the sample should look like after the supernatant has been removed properly. If you remove too much, yields will be lower, as you will likely remove a part of the leucocyte pellet. Leaving too much supernatant will lead to protein contamination and may interfere with DNA binding to the beads.
If you are working with several samples, close each of the caps after the supernatant removal to prevent the pellets from drying out.
The third thing to keep in mind is that it’s essential that leukocyte pellets are completely resuspended at each resuspension step for the optimal DNA yield and solubility. There are 2 ways that you can resuspend the pellet – you can vigorously flick the tube, or you can drag the tube forcefully along a tube rack. We find this to be the most effective approach.
After flicking or dragging to resuspend, vortex briefly, add the next buffer, vortex briefly again to mix, and check to ensure that the pellet is resuspended completely.
Complete resuspension is particularly important during nuclei prep and lysis. This is because the leukocytes have just undergone the stressful erythrocyte lysis procedure, so their viability is drastically reduced, which causes them to be stickier and more difficult to resuspend.
After the Nuclei Prep Solution is added and the sample is pipetted up and down a few times, there may still be aggregates of leukocytes that are not easily resuspended by just pipetting up and down. Pressing the pipette tip to the bottom of the tube while pipetting up and down applies additional force that helps to produce a homogenous nuclei solution. This will lead to optimal DNA solubility and the best results.
We hope that this video has been helpful. Check out some of our other videos for additional guidance on this and other protocols. Our technical support scientists are also always available to help – contact us at firstname.lastname@example.org.
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