Use of streptavidin-coated magnetic beads to obtain ssDNA
Posted 13 February 2009 - 05:58 AM
I am trying to purify 86 nucleotide ssDNA. Had anyone used dynabeads M-280 Streptavidin for ssDNA purification before?
I immobilized dsDNA (one strand with biotin, another strand with FAM) to the streptavidin bead.
I tried out two ways (heat and NaOH) to dissociate the non-biotinylated strand from the biotinylated strand following the suggestion given on the invitrogen product FAQ. However, when I run purified ssDNA on the agarose gel, I get a band at much larger molecular weight than the correct molecular weight of ssDNA.
Why is this so?
History of sample applied to dynabeads:
1) PCR reaction to obtain dsDNA
2) PCR product purification to remove primers (which is successful and produced a band on agarose gel at the correct molecular weight)
3) Apply purified PCR product to dynabeads.
Even if I do assymmetric PCR, standard gel extraction kit from Qiagen for instance will give very low yield for 86 nucleotide ssDNA (maybe 10-20%). Or is there any other method to obtain ssDNA after PCR?
Any help or suggestion will be greatly appreciated. Many thanks in advance.
Posted 13 February 2009 - 09:05 AM
Posted 14 February 2009 - 02:31 AM
Your ssDNA will not run in the same way as a dsDNA sample of the same length will on a non-denaturing agarose gel. ssDNA will fold and form complexes, with unpredictable results. You need to run the ssDNA on a denaturing gel. Easiest is a PAGE gel, which should give very good resolution at 86 bp.