PRINCIPLE

The EXTRACTME RNA & DNA KIT utilizes spin minicolumns with membranes which efficiently and selectively bind nucleic acids at high concentration of chaotropic salts. During the first isolation step, a tissue is homogenized in order to disintegrate intercellular bonds (epithelial tissue) and fragmentize high-molecular proteins (muscle or connective tissue). A homogenate is lysed with guanidine thiocyanate and detergents. Nucleases are inactivated by guanidine thiocyanate and β-mercaptoethanol (optional). The homogenate is separated from undigested tissue/cell that remains after centrifugation. First, DNA selectively bounds to DNA Purification Column. In the second order, RNA remaining in the filtrate bounds to an RNA Purification Column membrane in the presence of additional ethanol. A three-step washing stage effectively removes impurities and enzyme inhibitors. Purified RNA and DNA are eluted with the use of low ionic strength buffers and may be used directly in all downstream applications, such as PCR, qPCR, primer extension, sequencing, microarrays, Southern blotting, and in case of RNA also Northern blotting, RT-PCR, cDNA synthesis or stored until ready to use.

PRODUCT SPECIFICATIONS

SAMPLE MATERIAL

• fresh or frozen tissue: up to 30 mg
• tissue preserved in RNase inactivating buffers (e.g. RNAlater® , Ambion):
  up to 30 mg
• cell culture: up to 10⁷ cells

BINDING CAPACITY

~230 μg RNA, ~60 μg DNA

TIME REQUIRED

• 10 minutes (for simultaneous RNA/DNA purification, lysis and homogenization time not included)
• 15–30 minutes for homogenization in liquid nitrogen
• 15–20 minutes for mechanical homogenization (ceramic beads)

RNA/DNA PURITY

A₂₆₀/A₂₈₀ ratio = 1.9 – 2.1