The NEBlot® Kit is designed to produce labeled DNA probes of high specific activity suitable for use as hybridization probes for screening gene libraries (1), probing of Southern and Northern blots (2,3) and in situ hybridization (4).
Using the method of Feinberg and Vogelstein (5,6), random sequence octadeoxyribonucleotides serve as primers for DNA synthesis in vitro from denatured, double-stranded template DNA by the Klenow Fragment of E. coli DNA Polymerase I.
The method permits the labeling of small quantities of DNA (< 25 ng). The resulting extremely efficient use of labeled deoxynucleotide triphosphate allows probes of high specific activity to be produced. It also enables the labeling of DNA in the presence of low-melting point (LMP) agarose, thus overcoming the major limitations of earlier nick translation methodology.
The system produces consistent results; typically, probes labeled to > 1 x 109 dpm/µg can be achieved within 30 minutes using quantities of DNA from 25 ng to 2 µg. All four dNTPs are provided allowing the researcher flexibility in choosing label. The performance of the system may be monitored by labeling the control DNA provided with the system.
NEBlot Kit is tested for its ability to label 25 ng of HindIII digested lambda DNA to a specific activity of > 1 x 109 dpm/µg.