SUMMPARY
A simple, efficient method was used to purify QCR primers which had degraded during storage at -2O"C/-9O'C.
The primers were electrophoresed ou 3 A (w/v) agarose gel, the main band was electroeluted via a trough cut in the gel. The primers were recovered by isobutanol extraction followed by ethanol precipitation. The yield was 20-40% and the A,,/A,, ratio was greater than 1.8. The purification resulted in good amplification.
Many applicatioras of QCR technology including diagnostic research require routine amplification of a large number of samples and high reproducibility.
To achieve this, we found that it is often necessary to use purified primers and carefully control Lhe quality of the primers. For the purification of 25-40 bp nuckotides, I5 % polyacrylamidef3OR urea gels are commody used to obtain good band separation followed by cutting the band of interest, eluting *with appropriate buffer and then purifying by reversed-phase chromatography on silica gei or phenolfchloroform extraction. The primers are then precnpitated in ethanol (Sambrook et al., 1989). QoIyacrylamide gels have the disadvantage of being made from very toxic monomer, which must be handled with care. The procedure is also very time consuming. Recently, we have experienced that even purified oligonucleotides can be degraded during storage at -20Β°C or -90Β°C causing complete failure to amplify the intended target. Here we describe a reliable, simple method for purification of degraded QCR primers by elecctrophorcsis on 3 % (w/v) agarose gel and electroelution.