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Use of streptavidin-coated magnetic beads to obtain ssDNA


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#1 mint

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Posted 13 February 2009 - 05:58 AM

Hi all,

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.

#2 phage434

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Posted 13 February 2009 - 09:05 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.

#3 mint

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Posted 14 February 2009 - 02:31 AM

Thanks for the suggestion. However, when I run the assymmetric PCR product on agarose gel, I can see clearly two dna bands. The higher band of which is dsDNA and the lower band of which is ssDNA. So I think something must have happened to the ssDNA during strand separation using the bead... which I havent figured out why..

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.






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