Use of streptavidin-coated magnetic beads to obtain ssDNA - Use of streptavidin-coated magnetic beads to obtain ssDNA (Feb/13/2009 )
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.
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.
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..
phage434 on Feb 14 2009, 02:05 AM said:
I've made a little research about streptavidin-coated magnetic beads. I would like to immobilize biotinylated PCR products. I have 3 candidate products. Theoretically, all of these items are ideal for this purpose.
There is huge price difference between these products (and some differences in certain parameters).
- Dynabeads® M-280 Streptavidin ; bead diameter: 2,8 um ; Binding Capacity (free biotin): 650-900 pmoles/mg beads ; concentration: 10 mg/ml ; 10 ml : 1 500 $
- solulink ; bead diameter: 0,8 um ; Binding Capacity (free biotin): > 14 nmol/mg ; concentration: 10 mg/ml ; 10 ml: 800 $
- spherotech ; bead diameter: 0,5 - 4.5 um ; Binding Capacity (free biotin): no described ; concentration: 0,5 - 1 w/v % ; 10 ml: 200-250 $
But I don't know what so special in M-280 making it very expensive. :)
Can anybody tell me what I should choose ?
Thanks for the answer ! :)