PRINCIPLE

DNA purification procedure consists of five steps and utilizes spin minicolumns with membranes, which efficiently and selectively bind nucleic acids. In the first isolation step, red blood cells are lysed. The cells contain no DNA and are a potential source of PCR inhibitors. They must therefore be separated from the white blood cells prior to DNA isolation. Then the white blood cells  are subjected to enzymatic lysis by Proteinase K in optimized BL Buffer. At this stage, all cell  membranes and proteins are degraded. After the addition of chaotropic salts, lysate is applied to purification minicolumn membrane and DNA is  bound. A two-step washing stage effectively removes impurities and enzyme inhibitors.  Purified DNA is eluted using either a low ionic strength buffer (Elution Buffer) or water (pH 7.0–9.0) and may be used directly in all downstream applications such as PCR, qPCR, Southern blotting, DNA sequencing, enzymatic restriction, ligation and so forth, or stored until ready to use.

PRODUCT SPECIFICATIONS

SAMPLE MATERIAL

up to 1 ml of fresh or frozen blood, up to 200 μl plasma, serum, buffy coat, lymphocytes or body fluids

BINDING CAPACITY

Approx. 27 μg DNA

TIME REQUIRED

25 minutes (including incubation time).

DNA PURITY

A²⁶⁰/A²⁸⁰ ratio = 1.7 – 1.9

RECOMMENDATIONS AND IMPORTANT NOTES

Co-extracted vestigial RNA contamination may interfere with some enzymatic reaction, but does not inhibit PCR. If an RNA-free DNA sample is desired, add 4 μl of RNase A solution (10 mg/ml; cat. no. RP14, RP45) to BL Buffer in step 5 of the Isolation Protocol. Mix well and incubate at 37°C for 5 minutes. After incubation, follow the Isolation Protocol from step 6 (section XI).