The ReliaPrep™ Blood gDNA Miniprep System provides a fast, simple technique for the preparation of purified and intact DNA from mammalian blood. Samples are processed using a cellulose binding matrix with alcohol-free washes in a microcentrifuge tube format (1)
. Up to 200µl of blood can be processed per purification. The genomic DNA isolated is of high-quality and can be used in common applications such as agarose gel analysis, restriction enzyme digestion and PCR analysis. The high sample purity also makes it compatible with applications dependent on DNA quality such as genotyping arrays (2)
. Samples are typically eluted in 50–200µl of water; however, in this report, we seek to increase the concentration of the eluates by eluting in only 30µl of water without sacrificing purity. We also test whether eluting a second time by adding the first elution back to the column would increase yield and further increase DNA concentration.
Venuous blood was collected from a single donor into EDTA blood collection tubes and DNA extracted the same day. Two hundred microliters of blood was used per DNA purification. ReliaPrep™ Blood gDNA Miniprep System (Cat.# A5081) was used for the DNA purifications, following the standard purification protocol in Technical Manual #TM330 except as indicated. For the single elution, 30µl of water was used to elute the DNA. For the double elution, the eluate from the 30µl water elution, was added back to the column. After incubating 1 minute at room temperature, the column was centrifuged at maximum speed for 1 minute. Eluated DNA concentration and purity ratios were determined by absorbance measurements using a Nanodrop Spectrophotometer. Downstream performance was analyzed by multiplex qPCR, which includes both a primer set for quantitation and an internal PCR control to test for the presence of inhibitors (Plexor® HY System, Cat.# DC1001). Samples were diluted 1:100 in 1X TE buffer before use in the Plexor® HY System. A BioRad CFX96™ real-time thermocycler was used in conjunction with Plexor® analysis software for the analysis of the DNA.
Two experimental methods were performed with the ReliaPrep™ Blood gDNA Miniprep System. The single elution method used the standard elution procedure but using only 30µl of water, compared to the 50–200µl recommended in Technical Manual #TM330. The DNA concentrations in these eluates were determined by spectrophotometry (Table 1). With a single 30µl elution, DNA concentrations ranged from 69ng/µl to 125ng/µl, with an average of 111ng/µl, which is similar to recoveries reported for the standard 50µl elution (1)
. With a final recovered eluate volume of 28µl, this resulted in 3.1µg of purified genomic DNA.
The double elution method was performed using the same method as the single; however, the 28µl eluate was then added back to the column and eluted a second time. The final eluate volume did not change between the first and second elution and resulted in 28µl final volume. The final average concentration of the eight purifications was 161.9ng/µl, which was 46% more than the single elution (Table 1). The final average yield was ~4.5µg.
The purity ratios, A260/A280 and A260/A230, indicated good quality DNA for both single and double elutions. Real-time quantitative PCR data indicates a similar trend in concentration to that determined by spectrophotometric readings (Table 2). Cycle threshold (Ct) values and concentrations demonstrated that DNA was purified from both methods and able to be amplified. Internal controls in the Plexor® HY experiment were not significantly altered, indicating there were no problems with inhibitor carryover in the eluates from the ReliaPrep™ System.
The ReliaPrep™ Blood gDNA Miniprep System was used to purify genomic DNA from 200µl of blood and efficiently eluted DNA in as little as 30µl. The 30µl elution resulted in average DNA yields of 3.1µg and concentrations of 111ng/µl. Concentration and yield increased by 46% to 162ng/µl and 4.5µg, respectively, by reapplying the eluate to the columns for a second elution. Both elution methods tested gave high-quality DNA, as determined spectrophotometrically by the A260/A280 and A260/A230 ratios, and performed well in multiplex qPCR with no detectable PCR inhibition.