Monarch®HMW DNA Extraction from Cell & Blood: Protocol Overview
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In this video, we walk through the protocol for extraction of high molecular weight DNA (HMW DNA) from cultured cells.
In this video, we are going to walk you through the workflow for high molecular weight DNA extraction from cells using the Monarch HMW DNA Extraction Kit for Cells & Blood.
The protocol is broken into two parts, Cell Lysis and DNA Binding and Elution.
In the Monarch Workflow, the cell membranes are first lysed to enable the RNA in the cytoplasm to be digested by RNase A. The nuclei, containing the genomic DNA, remain intact.
If the nuclei were also lysed in this first step, the lysate would become very viscous from the genomic DNA, and this would prevent efficient RNA removal by RNase A enzyme.
After pelleting the cells, prepare the Nuclei Prep Solution by mixing the Nuclei Prep Buffer with the RNase A and prepare the nuclei lysis solution by mixing the Nuclei Lysis Buffer with the Proteinase K.
Flick the tube to resuspend the cell pellet and add 150 µl of the nuclei prep solution. Pipette up and down 10 times to mix, being careful not to introduce air bubbles. The sample will become less turbid, indicating that the cells are lysing, but the nuclei will remain intact. Then, incubate at room temperature for 2 minutes.
This nuclei prep process helps to reduce the viscosity of the lysate so that the RNase treatment can be effective.
Next, add 150 µl of nuclei lysis solution and invert 10 times to mix. Avoid introducing air bubbles, and do not vortex or pipette. In this step, the nuclei are lysed, releasing the genomic DNA.
Incubate at 56°C for 10 minutes in a thermal mixer with agitation at the desired speed to control the shearing and tune the size of gDNA.
The speed of the thermal mixer influences fragment length; higher speeds reduce overall size. For the standard ligation-based nanopore sequencing, agitation at 2,000 rpm is recommended. At 300 rpm or with no shaking, maximal fragment length, in the Megabase range, can be achieved. These samples will be highly viscous and difficult to process but will provide the longest possible DNA fragments.
During this incubation, It’s helpful to prepare and label all the plastics that you will need for part 2 of the protocol.
After the 10 minute incubation, add 75 µl of the Precipitation Enhancer and mix by inverting 8–10 times. This solution creates the optimal salt conditions for the DNA to precipitate and bind to the beads.
Using clean forceps, add 2 DNA Capture Beads to each sample, which at this point should be contained in a Monarch 2 ml Tube. Add 275 µl isopropanol, close the cap, and mix on a vertical rotating mixer at 10 rpm for 4 minutes to attach DNA to the beads.
If you don’t have access to a vertical rotating mixer, you can invert the sample manually 25-30 times, slowly and gently. A manual inversion is complete when the tube returns to the upright position, and each inversion should take 5 or 6 seconds. If you carry out the inversions slowly enough, no beads will stick to the bottom of the tube.
After a few inversions, the solution becomes more viscous and the DNA will wrap loosely around the beads. During the following inversions, precipitation of DNA may be visible, especially with larger sample inputs. The DNA complex will often contain small air bubbles and with more inversions, the DNA will completely wrap around the beads, often causing them to stick together. Once the DNA is completely wrapped around the beads, the viscosity of the solution will drop back to normal levels.
After the inversions are complete, remove and discard the liquid by pipetting, and there are two ways to do this:
The first option is to keep the tube upright and insert pipette tip, gently pushing the beads aside to remove the liquid.
The second way is to angle the tube so that the beads remain at the bottom, and liquid reaches toward tube opening. Then, pipette from the liquid surface and continue to tilt the tube as you remove the liquid.
Add 500 µl of gDNA Wash Buffer, close the cap, and mix by inverting 2–3 times. Then, remove the wash buffer by pipetting, using one of the methods just described.
Repeat this wash step and once again remove the buffer. This time, you can pour the buffer out using the pipette tip to ensure the beads stay in the tube.
Make sure the bead retainer is inserted into a collection tube and pour the beads into the bead retainer. Discard the used 2 ml tube. A quick pulse spin removes residual wash buffer efficiently, and there is no drying step necessary.
Separate the bead retainer from the collection tube, discard the collection tube and pour the beads into a new, labeled 2 ml tube. Insert the used bead retainer into a labeled low bind 1.5 ml microfuge tube – this will be used for elution.
Add 100 µl of elution buffer onto the glass beads and incubate for 5 minutes at 56°C in a thermal mixer with agitation at the lowest speed. Halfway through this incubation, take the sample out of the incubator and tilt the tube almost horizontally and shake it gently. This ensures that the beads can move freely, allowing for complete release of the DNA from the beads. Place the sample back into the thermal mixer and finish the incubation.
Make sure the bead retainer is inserted into the 1.5 ml microfuge tube. After the 5-minute incubation, pour the eluate and the glass beads into that bead retainer and close the cap.
Centrifuge for 30 seconds to separate the eluate from the glass beads. When taking the samples out of the centrifuge, remove and discard the bead retainer with the beads and close the microfuge tube containing your eluate.
Before measuring or using your high molecular weight DNA, you’ll need to ensure it is uniformly dispersed.
To do this, first pipette up and down 5-10 times with a wide bore pipette tip.
Next, you have three options to help the DNA return to its natural conformation in solution. First, you can heat at 37°C for 30 minutes to 1 hour. Alternatively, you can leave at room temperature overnight. The third option is to leave at 4°C for at least 24 hours.
Any time you want to quantify your DNA, it is important to homogenize the sample by pipetting up and down with a wide bore pipette tip in order to get accurate measurements. If your sample is stored at 4°C, spin down the sample before pipetting to collect any droplets that have formed in the tube.
We provide more detailed guidance on this online and in the product manual.
If you have any questions or need help with your preps, our technical support scientists are happy to help; contact us at firstname.lastname@example.org