The EXTRACTME DNA TISSUE kit is designed for the rapid and efficient purification of high quality DNA from solid tissues (fresh, frozen, formalin-preserved or paraffin-embedded), physiological fluids, hair, rodent tails, insects and cell cultures. The kit includes additional bead-beating tubes with ceramic filling for tissue soft homogenization. The isolation protocol and buffer formulations have been optimized for high isolation efficiency and DNA purity.
- fresh or frozen solid tissue: 1-30 mg
- formalin-preserved tissue: 1-30 mg
- paraffin-embedded tissue: 1-30 mg
- cell culture: 103-107 cells
- physiological fluids (urine, PMR, peritoneal fluid): 1-5 ml
- hair: 1-30 mg
- insects: 1-30 mg
The typical efficiencies of DNA isolation from fresh biological material are given in section XIII.
approx. 30 μg DNA
- approx. 12 minutes (lysis time not included)
- 30-40 minutes for mechanical homogenization
A260/A280 ratio = 1.7 – 1.9
The DNA purification procedure consists of four steps and utilises spin minicolumns with membranes which efficiently and selectively bind nucleic acids. In the first isolation step, the tissue is homogenized in order to disintegrate intercellular bonds (epithelial tissue) and fragmentize high-molecular proteins (muscle or connective tissue). Then the homogenate is lysed by Proteinase K in optimised TL Buffer. At this stage, all the cellular membranes and proteins are degraded. When a metabolically active tissue is used for isolation, RNA is removed by the RNase A. The homogenate is separated from the undigested tissue remains by centrifugation and combined with chaotropic salts. The mixture is then applied to the purification column membrane and the DNA is bound. The two-step washing stage effectively removes impurities and enzyme inhibitors. The purified DNA is eluted using a low ionic strength buffer or water (pH of 7.0-9.0) and can be used directly in all downstream applications such as qPCR, Southern blotting, DNA sequencing, enzymatic restriction, ligation and so forth, or stored until ready to use.
The quality of each production batch (LOT) of EXTRACTME DNA TISSUE PLUS kit is tested using standard QC procedures. The purified DNA concentration, quality and stability are evaluated by gel electrophoresis and spectrophotometer. In addition, the functional quality is tested by qPCR and digestion with restriction enzymes.
Manufacturer: BLIRT S.A.