DNA was extracted from three anaerobic Gram positive bacteria, (C.difficile,B.bifidium and B.lactis) using a QIAGEN kit. PCR was performed for 16S rDNA segment specific for each one. Only B.lactis gave the product expected. I checked the DNA of the three bacteria and DNA smears were prominent in all (attached the gel) . I tried to repeat the PCR for B.bifidium and C.difficile with a new DNA aliquout and more MgCl2 (3mM in total), but still no specific product appeared knowing that the size expected is just 157bp for C.difficile which as i know shouldn't be affected by DNA degradation. (attached a gel for gradient PCR for both bacteria (Tm 55-65).
What should i do to my DNA samples now to solve the problem, instead of going through bacterial culturing and DNA isolation once more? Why B.lactis DNA gave a product knowing that it's product size was around 400bp while the others did not?
Steve...I am using a DNA of 10ng/ul of the bacteria in target (positive control) for each specific primers. The PCR components are working with other PCRs with other bacteria with no problem. The electrophoresis conditions i used are a voltage of 100mV , current of 60mA for 35 minutes. Any suggestions?
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