Description
Hard-to-break samples including fatty and tough fibrous tissues (e.g., tendons or ligaments) pose significant challenges in the first step of DNA isolation. Conventional ways of sample rupture or enzyme digestion may not completely lyse those challenging samples. The SPINeasy® DNA Kit for Tissue (with Lysing Matrix) can efficiently isolate high-quality genomic DNA from various types of tissue in 30 minutes. Samples are rapidly disrupted by bead beating method along with Lysing Matrix M and Buffer TD1. It is highly recommended to use FastPrep® Instrument from MP Biomedicals to disrupt the tissue for an optimal yield. The lysis buffer mix containing Proteinase K can completely dissolve the sample in a short 10-min incubation. The combination of mechanical, chemical, and enzymatic lysing methods of this kit provides significantly higher yields of genomic DNA as compared to other commercial kits that have no mechanical lysis. With this optimized lysis method, the hard-to-lyse samples such as tough (rodent tail, ear punches), and fibrous (heart, muscle) tissues can now be easily and completely lysed. The lysis buffer mix also contains RNase A which will minimize the possibility of RNA contamination. The subsequent treatment with Buffer TD2 enables selective binding of DNA to Column S. The genomic DNA extracted from multiple types of tissues showed no inhibition in PCR and is immediately ready to be used for downstream applications, including long fragment PCR, qPCR, restriction digestion and sequencing.
Figure 1: Comparison of quality and quantity of gDNA extracted from various types of tissues using SPINeasy® DNA Kit for Tissue (with Lysing Matrix) and competitor Q kit. The gDNA yield, A260/A230 and A260/A280 ratio (from 15 mg of corresponding tissue types) were measured using a spectrometer. The virtual gel image and DNA Integrity Number (DIN, in green and yellow) were analyzed using Agilent Tapestation 4150.
Figure 2: Amplifiability of DNA extracted with SPINeasy® DNA Kit for Tissue (with Lysing Matrix)
Upper panel: Agarose gel electrophoresis of gDNA isolated from various rabbit/mouse tissues using SPINeasy® DNA Kit for Tissue (with Lysing Matrix). One microliter of extracted gDNA was amplified using rabbit or mouse-specific α-actin primers (1kb product). Reactions without DNA served as negative controls (NC).
Lower panel: Comparison of threshold cycles (Ct) of qPCR when amplification was performed using equal quantity of chicken liver, beef, and fish gill gDNA (25 ng). Targets were amplified with SYBR green.