Recently I have been trying to amplify the 16S rRNA gene with few primers already used in different studies. In my previous study I got the same bands with previously described methods, but this time I tried continuously with same procedures and even with some possible modifications like because of possible taq polymerase inhibitors in these kind of samples, I used all the possible dilutions of the sample (DNA) to avoid this problem but didnt succeeded. I even used gradient pcr to adjust the primer annealing but of no use. Again I tried different concentrations of taq and dNTPs and some stabilizer like BSA but still not successful. As for as DNA concentration is concerned I have check it on genequant instrument its better than what I used before with positive results. And remember as a normal pcr for cloning purpose I have already got the bands of my gene with other primers but through nested pcr technique . But for this step I cant use that method because I need these bands for QPCR to investigate abundance. Can anyone help me in this situation? Thanks
And the controls? That's the first thing to check the PCR works or not. If the positive control doesn't work, the problem is not in the samples. If it does, then you may have inhibitors, very low DNA concentration, or maybe you don't have enough DNA.
Check a PCR spiking some samples with a positive control (C+). COmpare the band with C+, if the band is weaker, you have inhibitors, if not, maybe something else. How much Taq did you try? PCR cycles? DNA? For my complicated samples I had to go up to 1 U Taq, 35 cycles and 2 uL of DNA to get something, and I even had to concentrate some samples that gave a barely visible band to get a decent amplification
thanks for valuable suggestions , i have already tried all the possibilities you said. but in some steps i got smears instead of bands. i know that desired gene is there in the samples as i said earlier that i have got bands with nested pcr technique, but i cant use for qpcr. infact sediments always have a problem with many inhibitors but we can only reduce this by making dilutions which i have already tested. do you think amount of dNTPs may effect the result , especially smearing , or absence of bands or band on start even before the first band of ladder used? thanks
Maybe try with another TaqPolymerase (get a free sample from a company). I had issues with PlatimumTaq from Invitrogen then i used a "cheap" Taq and it worked. Also maybe you should include a humic acid prewcipitation step in your DNA extraction they can get really nasty in soil/sediment extractions. (i think it was with Potassiumacetate)
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