I have extracted the DNA from waste water sample by using the Zymo research protocol. I have used 16S(47F, 1492R) primer to amplify DNA. If any suggestion I would be so grateful.
It could be due to the nature of DNA in your samples as it may be fragmented or the DNA could bind with protein
can you specify which kit please Sunil. If the final step is precipitation and there is not much DNA then a co precipitant of glycogen or rna will help the dna to come out of solution and may increase the yield
Hi Paul,
Final step: After the DNA is was with wash buffer it was then centrifuged, 100 µl DNase-Free Water was added directly to the column matrix, stand for 5 minutes,
and then centrifuge was carried at 16,000 x g to elute DNA. Zymo Reserach kit available at :http://www.zymoresearch.com/downloads/dl/file/id/700/d7003i.pdf .
Even if we have good quantity of extracted DNA, it did not show good band. Good quality bands depends upon DNA concentration and the gel you have made for gel-electrophoresis.
If more concentrated DNA then less amount of agarose gel will be used and vice versa. I recommend you to change the gel concentration. If you don't want to change then go for Nano-drop analysis, it will detect DNA even in nano-gram range.
You need to check whether DNA has some PCR inhibitory molecules. For that you can just add this DNA into any other template specific PCR using primers for that particular template. If the PCR amplifies without your DNA and does not amplify when you include this DNA, it means the preparation has inhibitory substance and you need to re precipitate DNA to remove such inhibitory substance.
did you measure the amount of dna present in the 100ul of eluted water/dna there may just not be very much of it. I would also add a little TE because I do not like storing dna in water. Water absorbs co2 from the air and the acid produced can depurinate dna eventually. Even if you cannot see dna it may still be worth trying PCR as that needs very little template
the yielded DNA was too low to be appear on gel the resolution of agarose gel depends on the DNA concentration>>> try to measure the concentration ... or culture the microorganism from your sample before DNA extraction ....good luck
Yashwant and Paul , PCR inhibitor removal kit was used and also concentration of DNA was measured.
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