Q. What’s the most effective tool/procedure for removing the salts, unincorporated primers, and other detritus from DNA preps prior to injection into the separation columns?
A. We have two preferred methods for cleanup of fluorescently-labeled di-deoxy sequencing reactions prior to loading on AB3100 or AB3730xl sequencing instruments.
1) For small numbers of samples (up to 24), the Princeton Separations’ CentriSep spin column is very effective. The processing time, including drying of the column eluate, is under 30 minutes. A bench top microcentrifuge is required. The method is reproducible and reliable.
2) For large numbers of samples, especially in 96- or 384-well microtiter plates, a simple ethanol precipitation followed by an ethanol wash is least expensive and gives good results. This procedure is technique-sensitive, requires a large centrifuge with buckets for multi-well plates and takes about 2.5 hours to complete. Compared to method 1, however, it is very time-efficient and cost-effective considering the many-fold larger number of samples processed.
Associate Investigator, Genome Technology Branch
Director, Sequencing Group, NIH Intramural Sequencing Center NHGRI
A. In our experience, ethanol precipitation is quite effective in removing salts, unincorporated primers and other debris from sequencing reactions prior to injection into our ABI 3730xl sequencers. However, while performing BAC/Fosmid end sequencing, where the amounts of ABI Big Dye Terminator are much greater than for small plasmids, ethanol precipitation is less effective in removing the dye blobs, which can lead to low quality sequencing regions. For such applications, the SPRI (solid phase reversible immobilization) clean up system from Agencourt Bioscience, called CleanSEQ, is very effective.
Arvind Kumar Bharti
The Plant Genome Initiative at Rutgers Waksman Institute
Rutgers, The State University of New Jersey
A. JGI uses a magnetic bead procedure for cleaning up sequencing reactions. We have found this method to be far superior to standard ethanol precipitation. The beads bind DNA but not protein or salts, so these are washed away and we end up with very pure product. In addition, we think that some of our template remains bound to the beads during elution, further purifying the product. In addition, the entire protocol is fully automated, meaning no centrifuges. JGI protocols can be found at: http://www.jgi.doe.gov/Internal/prots _index.html.
Paul M. Richardson, Ph.D.
Research and Development
US Department of Energy
Joint Genome Institute
A. We don’t do anything super-creative or “sexy” to clean up Big Dye terminator reactions prior to going on the gel. Ethanol precipitation is still the best way to achieve this — ethanol and salt with a spin of the plate to precipitate, dump that off and then add 70 percent wash, spin and dump. We speed-vac the plate and resuspend in water just prior to injection onto our ABI sequencers. This has scaled successfully to our current daily throughput of >167,000 sequencing reactions.
Assistant Professor, Genetics and Molecular Microbiology
Washington University School of Medicine, Genome Sequencing Center
A. The preferred and least expensive method for cleaning a PCR product is Exo/SAP (Exonuclease I + shrimp alkaline phosphatase). If you are sequencing, however, you must make sure that you only have one band in order to use this method. Alternatively, if multiple bands appear on your gel, you must run your entire PCR product (i.e. 25 to 50 µL) on a gel and cut your band of interest. Then you can use a Qiagen kit to extract your pure PCR product from the gel. After extraction, run 1 to 2 µL on a gel to make sure that you actually got some product and then submit for sequencing. As for cleaning up post-sequencing reactions, edge columns from Edge Biosystems or Sephadex G50 from Amersham will do the job just right.
UCLA Sequencing Core Specialist
UCLA Sequencing and Genotyping Core