Tips for Efficient Lysis of Tissue Samples Using the Monarch Genomic DNA Purification Kit

Tips for Efficient Lysis of Tissue Samples Using the Monarch Genomic DNA Purification Kit


Sample lysis is an important and often overlooked
step during the purification of genomic DNA. Cell membranes and extracellular matrix components
must be fully disturbed, and proteins and RNA must be completely degraded to ensure
gDNA is fully exposed and available for capture by the column. Lysis of tissue samples can be especially
challenging. In this video, we will provide some tips to
ensure the proper lysis of your tissue samples. The success of the lysis step is dependent
on input amounts, which differ based on the tissue type. Using excess material can overload the buffer
chemistry and clog the column, resulting in low yields and poor DNA quality. For example, DNA-rich tissues like spleen,
liver and kidney will become extremely viscous during lysis, which can prevent efficient
digestion of the tissue if recommended input amounts are exceeded. Please reference our recommendations for sample
inputs online to ensure that you use the correct amount. When you are ready to begin processing the
tissue, the samples should be cut into small pieces and processed in a 1.5 ml microfuge
tube. After adding Tissue Lysis Buffer and Proteinase
K, the samples should be mixed thoroughly. Make sure there are no tissue pieces sticking
to the bottom of the tube, as they will not lyse efficiently. If there are pieces on the bottom of the tube,
vortex the tube to suspend them. When lysis is carried out using a thermal
mixer, it is usually complete within 30-60 minutes. You can proceed from here with the purification
procedure and you will have excellent results. However, if time isn’t limiting, lysis can
be extended for up to 3 more hours to obtain even higher yields and the lowest possible
RNA content. If you do not have a thermal mixer, you can
use a heat block. Vortex the sample every 20-30 minutes to speed
up lysis. Lysis is complete when the tissue pieces are
gone. RNase A should be added at end of the lysis
step, to prevent it from being degraded by the proteinase K. By the time lysis is complete, the RNA is
released from the tissue and is readily accessible for enzymatic degradation. When working with fibrous or fatty tissues,
or with samples stabilized with RNAlater, free floating insoluble fibers will form during
lysis, producing a turbid appearance. If these fibers are not removed prior to loading
the lysate onto the column, they will stick to the membrane and prevent DNA binding, effectively
reducing yield and quality. To remove the fibers, centrifuge the lysate
for 3 minutes at maximum speed and transfer the supernatant to a fresh tube before adding
binding buffer. For some of these tissues, the recommended
input amounts are lower in order to minimize fiber formation. If these inputs are exceeded, it may not be
possible to sufficiently remove them. 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.

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