What the best way to separate the single stranded DNA from a sample containing both ssDNA and dsDNA?
dear adel read more in this link
https://www.ncbi.nlm.nih.gov › NCBI › Literature › PubMed Central (PMC)
run your sample on the gel it will separate it for u. u can then extract single stranded dna by doing electroelution.
if this is pcr product then having biotin on one strand primer allows binding of the product to streptavidin beads then strand degradation with NaOH when immobilised then release of the bound single strand from the bead if the other strand is needed
there are more one method..
1-denaturing agarose :by mix 1 ul of PCR product with (3 ul) of Single strand DNA loading buffer using( EDTA, &mmNaOH) incubation for 5 min. at 90 c then rapid cooling, ,,,then electrophresis on agarose (2g- 5x TBE). x-ray film for 3-4 hours at( -80c ).
2-two-dimensional single strand dependent electropheresis(2D-SDE):
this method used to investigate a complex sample of nuclic acid(ssDNA, ds DNA, or may also RNA DNA hybrids):the protocol based on two electropheresis
1st is the non-denaturation electropheresis: separate the ssDNA from dsDNA from hybrids depending lenth and conformation
2nd is the denaturation electropheresis : using urea . this will separate all doubble strands and make all single strand>>>>with presence of one of labeling agents the single strand originate from dsDNA will apear as an arc behind original ssDNA.
see the following discussion on RG
https://www.researchgate.net/post/How_do_I_separate_ssDNA_and_dsDNA_at_70_C_without_destroying_either_one_of_them?view=54c37cc2d5a3f2c9728b464e
You can use online calculators. Here is the link- https://nebiocalculator.neb.com/#!/dsdnaamt
http://cels.uri.edu/gsc/cndna.html
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