Hello,
So I performed a DNA extraction (phenol-chloroform method) of still unkonwn strains (bacterial but could be archaeal), and after a positive quality check using nanodrop mesures (260/280 = 1.9 - 2.0), the concentrations were deluted to achieve a 100 ng/ul per samples.
Two PCR were performed:
- Targeting bacterial ITS sequences : from 17 samples, 15 worked : the usual aspect of profiles, some however showed only a sinlge band.
- Targeting 16S rDNA sequence : only 2 worked from the postive 15 samples (most of which are those showing a single band in ITS amplification).
(Check images)
What do you think could the reason be?
@Dirk Schmidt
Thank you for your answer. In that case I will try a series of dilutions, i.e. 50 ng/ul, 10 ng/ul and 1 ng/ul to see which would work the best.
As for the primers, the concentration used is 25 uM and I used these :
* for ITS :
- Forward = GTC GTA ACA AGG TAG CCG TA
- Reverse = CAA GGC ATC CAC CGT
* for 16S :
- Forward = AGA GTT TGA TCC TGG CTC AG
- Reverse = CTA CGG CTA CCT TGT TAC GA
Takwa Wannassi
No they are the standard primers as mentioned in my previous reply.
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