single stranded DNA purification using streptavidin agarose bead

[icey26]

Hi all,

Did anyone do single stranded dna purification using streptavidin agarose beads before?

I PCR amplified a single stranded dna library to get double stranded dna using reverse-biotinylated primer and non-biotinylated forward primer (doing symmetric PCR).

However, when I do the strand separation to get single stranded dna (i.e. immobilisation of dsdna to streptavidin bead, then use NaOH to denature the dsdna and collect the single stranded dna in the supernatant), I get single stranded dna that is of a much higher molecular weight (than the dna library) as visualized on the gel. The dsdna is at the right position on the gel.

I don't know why this is so and no one else seems to have any problem using streptavidin beads to get single stranded dna... I presume as I can't find any troubleshooting guide with regards to this aspect...

Please help!! I don't know why a technique that is so widely used cannot be done by me..:-(. Any suggestions will be greatly appreciated.

[markyan]

Hi Sir,
I also use the streptavidin bead to isolated the ssDNA after NaOH treatment. The procedure works fine with me. However, the ssDNA yield is very low in comparison to the amount of dsDNA I incorporated into the system. As the size of the ssDNA is way too large, I suggest that you check the quality of the dsDNA you used for the NaOH melting.
Thanks
Mark

[ashleyj722]

Originally Posted by icey26

Hi all,

Did anyone do single stranded dna purification using streptavidin agarose beads before?

I PCR amplified a single stranded dna library to get double stranded dna using reverse-biotinylated primer and non-biotinylated forward primer (doing symmetric PCR).

However, when I do the strand separation to get single stranded dna (i.e. immobilisation of dsdna to streptavidin bead, then use NaOH to denature the dsdna and collect the single stranded dna in the supernatant), I get single stranded dna that is of a much higher molecular weight (than the dna library) as visualized on the gel. The dsdna is at the right position on the gel.

I don't know why this is so and no one else seems to have any problem using streptavidin beads to get single stranded dna... I presume as I can't find any troubleshooting guide with regards to this aspect...

Please help!! I don't know why a technique that is so widely used cannot be done by me..:-(. Any suggestions will be greatly appreciated.

If your running a ssDNA on a non denatured gel then it will migrate slower on the gel compared to the dsDNA. This is because of the shape of the ssDNA