I am separating ssDNA from dsDNA using streptavidin bead. I electrophoresed the sample after separation on agarose gel. I can see a band of 1.7kb (dsDNA) and a band of 1kb. I am not sure whether the 1kb band could be my ssDNA?.
i have no experience with Streptavidin beads but you could consult this paper and author : "Nucleic Acid Ther. 2011 Dec;21(6):437-40. doi: 10.1089/nat.2011.0322. Epub 2011 Nov 2."
all the best
why are you getting any DSDNA Shalini?. If you are pcr-ing with biotin primer and denaturing any unincorporated biotin primer before binding to the bead then you should be able to get only SSdna off the beads. What denaturation method are you using for this please?. You have to consider that both of these band may be different conformers of the SS DNA, If you have a good amount of the large band you could try a restriction enzyme digets which would work on DS but not on SS dna with part of it. SS dna does run differently from DS dna as is usually seen in the SSCP mutation detection method and usually runs apparently larger. If you do try this you may want to run DS pcr product cut and uncut as controls just to check fragment sizes
I pcr-ed using biotin anti-sense primer. Gel extracted the amplified PCR product and denatured them with 30mM NaOH for 10 min before binding to the beads.
Is that right Shalini that you denatured the product before binding to the beads?
Would it be better to remove all unused primers then bind the DS product to the beads and then denature off the SS non biotinylated strand with NaOH while the biotin strand remains bound to the bead
Finally, just followed the Streptavidin Dyna bead protocol for ssDNA seperation (https://tools.thermofisher.com/content/sfs/manuals/dynabeads_myone_savC1_man.pdf) and electrophoresed the sample containing NaOH WITH OUT neutralising with HCl. It worked brilliantly and was able to extract enough ssDNA from the gel.
The other best method for generating long ssDNA is RT PCR using TGIRT-III.
However, the easy method for extracting ssDNA >4kb from dsDNA plasmid is by double digestion with nicking enzymes. The digested plasmid was directly loaded in denaturing gel loading buffer and electrophoresed on non denaturing gel with ethidium bromide. The correct size band was gel extracted.
You can design your own insert with nicking sites or clone it in a plasmid from commercial source (http://www.biodynamics.co.jp/images/news/ds610.pdf) which also provides denaturing loading buffer.
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