As a possible improvement to Tom's approach, do a preceding PCR with a much larger product by using an upstream forward primer.
That way, your biotinilated primers will bind halfway along the template, become extended to the length of the preceding PCR, but any remaining bound template will be easily separated on a gel when it comes to purification whereas single-stranded DNA will appear as a lighter band. i.e: PCR1 on raw template: F1> ============================ <R1 Product: F1===================R1 PCR2 with preceding product as template: [Bt]-F2> F1===================R1 SS Product size Vs. Template size Vs Product/Template Hybrids; F1===================R1 F1-------[Bt]-F2=========R1 - Biotinylated product bound to template [Bt]-F2----------------R1 The mobility of single stranded DNA vs. semi-single-stranded DNA versus double-stranded DNA, I don't know about. On the one hand, ssDNA has the same charge proportionally to its mass, but its length (and likelihood to be obstructed by gel fibres) remains the same. Hopefully someone more experienced in ssDNA gels can jump in here. Alternatively, you might be able to denature your DNA using alkaline conditions. I suspect that's what the ammonium hydroxide approach is intended for. Whether this will degrade the sepharose, break the biotin bond or otherwise screw up the DNA is beyond my ken to predict at present. I imagine it'd work, and from what I have seen there are ways of tuning your alkalinity precisely to specific sequences so that double-strands are broken based on their G-C content, etc. Perhaps you can keep us posted on your progress here, I'd like to know which method you use and which works! On 12 April 2010 06:57, Tom Knight <[email protected]> wrote: > > Guo Wei Kua <[email protected]> writes: > > > > I have PCR products of about 70bp. One strand is biotinylated, > > because I used biotinylated forward primers. Now, how do I isolate > > only this strand? Do I denature first, then use beads to capture > > the ssDNA? If so, how to do that? I am afraid that once I denature > > the dsDNA with heat, I won't be fast enough to capture the ssDNA > > before reannealing. Or do I capture the dsDNA first, then > > heat-denature the dsDNA so that the non-biotin strand will fall off? > > The problems hereby is, again, reannealing of the DNA. Second, > > heat-denature may even break the biotin-bead bond, causing > > everything to be lost when I discard the sup? Help!! > > > > PS: I prefer not to use high salt solutions, as the final purified > > ssDNA should not hv salt if possible. > > I think this is the difficult approach. Why not create just the > strand you want with linear amplfication using a single primer. Add > just the biotinylated primer to a "PCR" reaction with large amounts of > template DNA (perhaps a previous pcr with both primers, diluted > significantly, say 100x). Do cycling as normal, but expect linear > amplification with production of only the primed strand. Done, unless > you really need to eliminate ALL of the reverse strand. Otherwise, > purify with biotin. With this method you could probably work without > biotinylated primers at much lower cost. > > _______________________________________________ > Methods mailing list > [email protected] > http://www.bio.net/biomail/listinfo/methods -- letters.cunningprojects.com twitter.com/onetruecathal twitter.com/labsfromfabs _______________________________________________ Methods mailing list [email protected] http://www.bio.net/biomail/listinfo/methods
