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PCR contaminant - (Mar/27/2006 )

hi! im trying to amplify DNA from seaweed.. but it seems that the DNA template is still with polysaccharides...i cant amplify!!! i had tried diluting the DNA but still no pcr products...
i guess my extraction was successful because when i run my extracts through the gel, DNA were with high molecular weight... i used the xanthogenate method of dna extraction.
how could i possibly optimize my pcr? thanks...

-Haenz-

Are you sure your primers are okay?

Maybe try an isopropanol precipitation (add 0.1 volume of 3 M sodium acetate, mix well, then add 0.6 volumes of room temperature isopropanol)? You'll want to keep the alcohol content low, as polysachharides will ppt in alcohol, too. Here's another idea...

Maybe a phenol::chloroform extraction, followed by an alcohol precipitation?

-HomeBrew-

Do you have any source to confirm the primers you're using are ok? Any other possible form of control?

-vairus-

QUOTE (HomeBrew @ Mar 28 2006, 12:55 PM)
Are you sure your primers are okay?

Maybe try an isopropanol precipitation (add 0.1 volume of 3 M sodium acetate, mix well, then add 0.6 volumes of room temperature isopropanol)? You'll want to keep the alcohol content low, as polysachharides will ppt in alcohol, too. Here's another idea...

Maybe a phenol::chloroform extraction, followed by an alcohol precipitation?



thanks...:-) my primers are okay... my positive control is okay... that's why the problem is on the dna sample...

i'l try to precipitate the DNA with high salt... do u have a copy of the full text of the article from biotechniques? i only saw the abstract and i think it would be better if i see the full protocol.

would you know how much or how little alcohol can i use to precipitate the DNA and not the polysaccharides?

thanks...

-Haenz-

Hi

You could try a Phenol, chloroform, isoamyl alcohol extraction, it is supposed to lower the amount of polysaccharides extracted with the DNA.

-bob1-