- Gene Expression
- Northern/Reverse Northern
GenHunter offers several products for Northern & reverse Northern blotting:
- the HotPrime DNA labeling Kit (Cat. No. H501) for optimized labeling of differential display fragments with radioactively labeled dATP.
- the ReversePrime cDNA labeling Kit (Cat. No. R701) for labeling cDNA probes for use in "Reverse Northern" blots.
The HotPrime DNA labeling kit features three unique improvements over the traditional "random priming" method which allow for radioactive labeling of DNA probes with at least 5-10X more specific radioactivity. It is designed to efficiently label cDNA probes isolated from differential display for Northern or Southern blot analysis as well as library screening. However, the HotPrime Kit can also label DNA probes for any other application to a similar high specific activity. The first improvement over traditional random-prime kits involves using random decamers instead of hexamers. This ensures more efficient priming of DNA probes of any sequence based on the observation that primers shorter than 9 bases are inefficient in initiating DNA synthesis (Williams et al., 1991, Nucleic Acids Res., 18:6531-6535). Second, because the cDNA fragments from differential display are relatively short (150-700 bp), the incorporation of anchored oligo-dT primers into the labeling buffer ensures the "full length" anti-sense cDNA probes to be labeled, which greatly increases the chance for signal detection on the Northern blot or library screening. Third, the use of radioactive dATP instead of dCTP allows labeling of DNA to the highest specific activity by taking the advantage of the AT rich nature of the 3' untranslated regions of mRNA detected by differential display.
GenHunter's ReversePrime Kit provides a complete system for labeling cDNA for use in "Reverse Northern" blots to conduct differential screening of positive clones generated by differential display. This kit is ideal for either dot blot or colony hybridization screening of differential display PCR products cloned into the PCR-TRAP® Cloning System (Cat. No. P404). This improvement significantly reduces the amount of labor required for screening by Northern Blots or Ribonuclease Protection Assays and provides high specificity. It accurately identifies positives from false positives and reduces the amount of RNA needed to conduct differential display screening.