The phenol extraction of nucleic acids has always been an unpleasant element of any molecular biology protocol. The Phase Lock Gel (PLG) from 5 PRIME delivers an elegant solution to this inconvenience. During centrifugation, the PLG forms a barrier between the aqueous and organic phases. Thus, the water-soluble phase becomes well separated from the organic and interphase elements, allowing for the easy collection of DNA or RNA without the problem of cross-contamination with traces of organic phase. Because you don’t have to worry about organic phase contamination, PLG increases the yield of DNA by an average of 30%. PLG is available predispensed in centrifugation tubes that range in volume from 1.5 ml to 50 ml. It comes in two forms, PLG Heavy and PLG Light, for RNA and DNA extractions, respectively.
The first time we used PLG was for expression profiling experiments using microarray technology. The microarray platform we were using was the GeneChip from Affymetrix. For these experiments, high quality RNA, and subsequently synthesised cDNA, is essential to obtain conclusive results. For probe preparation, total RNA has to be reverse transcribed and cDNA purified for subsequent in vitro transcription. We observed that purification of cDNA using PLG singnificantly increases DNA yield. We have been also trying DNA purification columns from another manufacturer in order to avoid phenol/chloroform usage in our protocol. However, we clearly obtained a lower rate of cDNA recovery with these columns compared to a purification protocol based on PLG. This became even more evident in the case of RNA amplification protocols, where the amounts of cDNA subjected to a first round of in vitro transcription are extremely small. Another positive element about Phase Lock Gel is its moderate price in comparison to purification columns.
PLG is especially useful when small amounts of DNA have to be recovered. Normally, the risk of contamination is quite high since one tries to pipet up the maximal volume of aqueous-phase supernatant. One thing that can be a bit disturbing about PLG is the green color of the safe-lock tubes in which it comes. To a certain extent, the color of the tubes reduces the visibility of the phases and thus aspiration of the aqueous phase.
Nonetheless, I recommend the Phase Lock Gel to anybody – especially those that routinely perform phenol/chloroform extractions when purifying nucleic acids. PLG makes this part of the protocol much safer and more convenient.
Michal Janitz, M.D.
Max Planck Institute for Molecular Genetics
Berlin, Germany
At the time of this review Phase Lock Gel was commercialized by Eppendorf. Today it is now available from 5 PRIME. This review was edited to reflect this fact.