Downstream applications utilizing PCR products or restriction digested DNA often require that the nucleic acids are free of contaminating primers, proteins, and buffer constituents. Many kits are available to perform this function and are collectively referred to as PCR cleanup kits. Many of these kits employ a spin column or nucleic acid binding resin to hold on to the nucleic acid while the contaminants are removed by washing. However, in an effort to simplify the cleanup procedure even more, there are some kits that have eliminated the use of spin columns or resins and rely on a precipitating reagent to pellet the nucleic acids away from contaminants. I just tried a new product from Bioline (Randolph, MA), called SureClean Plus, that falls into this columnless/resinless format. Considering how easy the procedure was to carry out, the results were outstanding.
The SureClean Plus kit consists of 2 reagents, the SureClean reagent and a pink co-precipitant. The co-precipitant is optional but most likely would increase the yield from reactions containing small quantities of nucleic acid. The protocol is very simple. For a standard PCR cleanup, add 6 ul co-precipitant, mix well, add an equal volume of the SureClean reagent and mix. Let the mixture sit at RT for a minimum of 10 min, then pellet the nucleic acid in a microfuge, aspirate the supernatant, wash the pellet with 70% ethanol, allow the pellet to air dry and re-suspend in water or TE. It is, essentially, a standard precipitation protocol but much quicker and, from the results, much more efficient.
I tested the SureClean Plus kit on two 15 ul aliquots of a PCR reaction containing a 389 bp amplicon. Since most of the primers had been used up in the PCR reaction, I supplemented one aliquot with 20 pmol of a 20 bp primer before the cleanup in order to gauge the effectiveness of primer removal. After performing the cleanup as per the instructions, I ran the entire amounts of recovered material on a 1.5% agarose/0.25% Synergel TBE gel. In order to estimate recovery, I also ran a 15 µl aliquot of the original PCR reaction that had been supplemented with 20 pmol primer.
In the gel pictured, lane 1 contains the original PCR product, and lanes 2 and 3 contain the SureClean Plus-treated primer-supplemented PCR product and unsupplemented PCR product, respectively. It is obvious from the gel that the SureClean PCR cleanup 1) achieved essentially 100% recovery of the PCR product, and 2) effectively removed the added primer (the primer is more distinct in the original photo). For this test, I used the co-precipitant. Although I can’t say if it was necessary, the pellet was very easy to see.
Bioline’s SureClean Plus PCR cleanup kit is an excellent product, at least as far as simplicity, recovery, and, primer removal goes. Since I haven’t used the cleaned up DNA for any downstream applications yet, I can’t attest to the purity of the nucleic acid. However, since the reason for doing a cleanup in the first place is for downstream applications, my impression is that the purity would not be an issue. In addition to ease of use and effectiveness, SureClean Plus is also very economical. The 5 ml package size, containing 0.8 ml co-precipitant, sells for $95; the 25 ml size with 4 ml co-precipitant is $395. Assuming a standard PCR reaction is 50 ul, the smaller package size can perform 100 cleanups ($0.95/reaction) and the larger size can do 500 ($0.79/reaction). In contrast, a leading spin column-based PCR cleanup kit is about twice that cost.
Michael Campa, Ph.D.
Assoc. Research Professor of Radiology
Duke University Medical Center