Post-lysis Tips for Optimal Results using the Monarch®Genomic DNA Purification Kit
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After lysing your sample during your DNA extraction workflow, follow these tips to ensure that your purified gDNA is high-quality, intact, and free from contaminants.
After lysis, a sample is processed by a familiar bind-wash-elute workflow to purify the genomic DNA. In this video, we’ll share some important things to keep in mind when taking your lysed sample through these steps. Following these guidelines, in addition to our storage and lysis guidelines, will help you achieve excellent yields and highly-pure genomic DNA. When loading your sample onto the column, it’s important to pipette it directly onto the membrane and avoid transferring buffer components to the upper area of the column, as these could later carry over into your eluate. Keep in mind the following when loading your sample onto the column: Do not transfer any foam that may have formed during lysis; Avoid touching the column reservoir with your pipette when loading the sample. Close the cap gently and take care when moving the samples to and from the centrifuge to avoid splashing, which can transfer buffer components to the upper column area. You may notice that our binding step is carried out at two different centrifugation speeds – first at 1000g and then at maximum speed. Spinning at a low speed gives the DNA more opportunity to bind to the matrix. Then, spinning at maximum speed clears the membrane of remaining salts and proteins. We recommend that the columns be placed into the centrifuge in the same orientation, for example with the hinge pointing outward. This ensures the liquid takes the same path during each spin, optimizing purity and improving yields by around 5-10%. After adding the wash buffer to your samples, invert the column a few times or vortex briefly. This will aggressively wash away any remaining buffer salts that may have contacted the upper column area. After the first wash spin, empty the collection tube and briefly tap it upside down on a paper towel in order to reuse it in the next wash. This reduces the total amount of plastic used in the prep. The Monarch kit employs a warm elution step where the column is incubated for 1 minute with elution buffer that is pre-heated to 60°C. Pre-heating the buffer increases the efficiency of the elution, ensuring 80-85% of the genomic DNA elutes from the column in a single step. The use of a warm elution buffer increases the yield, when compared to a room temperature elution. Ensure that elution buffer is 60°C; cooler buffer will reduce efficiency and thus yield. If the buffer is above 60°C, the DNA may be partially denatured. As heating blocks may not be accurate, we suggest confirming the temperature of your heating element by filling a tube halfway with water and inserting a thermometer. It can also help to warm the pipette tip prior to transferring the elution buffer onto the membrane by pipetting up and down a few times with the warm buffer. The volume of the transferred elution buffer will be slightly larger than 100 l when using a pre-warmed tip. Alternatively, you can use elution buffer warmed to 70 degrees with a room temperature pipette tip. We recommend elution with 100 l, but you can reduce the elution volume to as low as 35 l if you’d like to obtain a more concentrated sample. However, this will lead to a reduction of the total yield of 20-25%. The supplied elution buffer contains 10 mM Tris, 0.1 mM EDTA at a pH of 9.0. The high pH and the EDTA give excellent protection against nucleases, which makes it highly suitable for long term storage at 4, -20 or -80°C. Alternatively, nuclease-free water or TE buffer can be used for elution. We hope that these tips have been helpful. If you have any questions, our scientists are ready to help. Contact us at info at NEB.com.