HTP PhyTip PCR Clean-up Columns

PCR reactions generate DNA fragments of interest. However, the reaction product also consists of contaminants that include excess dNTPs, primers, salts and failure products. In a typical cleanup, the target double stranded DNA may be 2kb in length.  PCR reactants and byproducts of 100 bp and smaller are eliminated. Yield and functionality are maintained while benefiting from high throughput automation.  The purified PCR products are suitable for generating RNA by reverse transcription, cloning, hybridization reacts, and a host of other applications.

PhyNexus utilizes a proprietary chemistry to clean up PCR products in a fully automated, 96-at-a-time format. Depending on the sample, PhyNexus employs a number of different chemistries for performing automated PCR clean up. These include ion pairing and chaotropic precipitation. Gel filtration and ion exchange may also be considered. The chemistries are focused towards not only optimizing the method to minimize sample loss, but on the downstream application desired. Sample size and automated throughput are also considered.

PCR product samples are purified with PhyTip silica columns using an chaotropic separation mechanism.  PCR products from 4 different amplicons were studied.  For each amplicon, 200 µL of samples were purified and 100 µL of purified product recovered.  The results are shown on the gel to the right.

Samples A, B, C, D, and E are differently sized amplicons.  Sizes are shown with the ladder to the left.  The 10 bp loading dye indicator is a reference marking the end of the gel.  The control samples on the right show unpurified PCR product.  The recovered purified samples are shown by the left columns.  The result show that the PCR products recovered remain concentrated and are not diluted by the process.  

The sample UV absorbances were measured with a NanoDrop spectrophotometer.  The unpurified controls (starting samples) recorded an A260/A280 ratio of 1.79-1.92 and an A260/A230 ratio of 1.26-1.47 indicate the presence of contaminants including salts and enzymes.  The purified PCR products record an A260/A280 ratio of 1.8-2.0 and an A260/A230 ratio of 2.0-2.2 indicating pure DNA. 

PCR Clean-up using PhyTip Silica Columns-chart