PRINCIPLE

DNA purification procedure consists of four steps and utilizes spin minicolumns with membranes which efficiently and selectively bind nucleic acids. Swab or semen sample is subjected to enzymatic lysis by Proteinase K in SSL Buffer. When isolating from semen, DTT must also be used. In this step, cell walls, membranes and proteins are degraded by lysis buffer and  Proteinase K. After 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 with then use of a low ionic strength buffer (Elution  Buffer) or water (pH 7.0-9.0) and can be used directly in all downstream applications such as PCR, qPCR, Southern blotting, DNA sequencing,  enzymatic restriction, DNA ligation and so forth, or stored until ready to use.

PRODUCT SPECIFICATIONS

SAMPLE MATERIAL

Buccal, nasal, pharyngeal, vaginal, blood and saliva swabs or semen.

BINDING CAPACITY

Approx. 25 μg DNA

TIME REQUIRED

45-50 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 the RNase A solution (10 mg/ml; cat no. RP14, RP45) to SSL Buffer in step 2 of the Isolation Protocol. Mix well and incubate at 37°C for 5 minutes. After incubation, add Proteinase K and DTT when isolating from semen and continue isolation following the Isolation Protocol (section XI).