I am using 1% CTAB for extracellular DNA extraction, followed by isopropanol precipitation, phenol chloroform extraction, ethanol precipitation and wash. However, the final eDNA yield was only about 3% (calculated by plasmid internal standard method from qPCR). And I think the key problem may occur in the CTAB precipitation step (50 mM Tris10 mM EDTA1% CTAB): qPCR shows that after precipitation and centrifugation, most plasmid is still in the supernatant, resulting in the low yield of eDNA. How can I increase my yield?
You could add a co precipitant such as linear acrylamide or glycogen in the precipitation steps to maximise the precipitation of the purified dna but possibly one answer is to do fewer purification steps and use a polymerase that is not too fussy about the quality of the DNA.....Kapa do a wide range of polymerases that work on less than ideal templates
3% of what? You can't know how much DNA you started with. Or did you include a known amount of plasmid DNA and see how much you recovered?
When you say "supernatant", which step are you referring to? Are you repeating the spin & remove aqueous layer until the upper phase is clear?
You can let the "precipitate -20*C in isopropanol" step go overnight, gives a better yield.
Have you checked to verify the temperature of your heat blocks?/water baths? If these aren't set properly, then you get a lower yield.
Checked the centrifuge settings to make sure it's spinning fast enough? You can increase the spin time to pellet the DNA. Can't really spin it too hard or too long. Small things can make a huge difference!
Good luck!
You could also consider concentrating the dna from a larger volume using speedvac or spin column concentration before doing the precipitation step
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