New England Biolabs, T3051L, Monarch© RBC Lysis Buffer

Related Categories High Molecular Weight DNA Extraction,, Nucleic Acid Purification FAQ Q: Can the Monarch HMW DNA Extraction Kits be used to process human Buffy coat? A: Yes, the Monarch HMW DNA Extraction Kit for Cells & Blood (NEB #T3050) can be used for processing Buffy coat. When working with Buffy coat, which is a concentrated PBMC solution, smaller input amounts should be used than when working with blood. The total cell count should not be higher than 1 x 107 cells for the standard protocol (agitation at 2000 rpm) and not higher than 5 x 106 cells when UHMW DNA is needed (low agitation). Buffy coat as obtained from blood banks contains up to 1 x 109 PBMCs per 30-80 ml, which equates to approximately 1 x 107 -3 x 107 cells/ml. We suggest starting with 100-300 μl of this solution, add PBS to a total volume of 500 μl, and move into the erythrocyte lysis procedure as if it were a 500 μl blood sample. Q: My blood sample is frozen in aliquots larger than is recommended in your protocol (e.g., in Vacutainers). Can I still use the frozen blood protocol for the Monarch HMW DNA Extraction Kit for Cells & Blood (NEB #T3050)? A: Yes, but it is important not to thaw the sample completely (or warm up) when it is not in the presence of RBC Lysis Buffer, as allowing the sample to warm will quickly activate nucleases. Incubate the blood container at room temperature until it is just about to start thawing (the color will become darker red). Place the tube in a tube rotator set at 20 rpm and rotate until the sample has almost thawed completely. Be sure to monitor the sample on the rotator very carefully to catch the point at which it is almost thawed. At that point, vortex briefly to finalize thawing and dissolve any aggregates and quickly place on ice. Take an aliquot of blood for your prep (recommend starting volume is 500 µl) and place in a 2 ml tube. Add 3 volumes of cold RBC Lysis Buffer, mix by inversion 5 times, and then continue with the frozen blood protocol (Step 5). Thawed blood samples should not be re-frozen. Alternatively, you can thaw the blood sample ever so slightly in a 37°C water bath, just enough to dislodge it from the container and move it into a larger container capable of accommodating the addition of 3 volumes of cold RBC Lysis Buffer. Then, the sample can be thawed in the presence of RBC Lysis Buffer according to the protocol. Please note that this approach may require additional RBC Lysis Buffer (NEB #T3051L).