qPCR using random primers - (Mar/27/2007 )

I wish to detect any kind of DNA in my sample. And the obvious thing that came to my mind is what if I use random primer (Hexanucleotides) as my primers. I felt they would aneal at different places irrespective of the sequence and amplyfy giving some signal which is enough for me to detect the presence of DNA. However, it didnt work and I failed to get any signal. I use SYBR green assay in ABI 7700SDS model. I have tried different primer concentration (from 20pmole to 200pmole); Different annealing temperatures (from 12 to 55 degree); different concentration of DNA in positive control.

My queries are:
1) why did i not get any amplification?
2) Is there a way where i may increase the probability of getting a signal?
3) any alternate strategy for similar detection which i may try?

Would be eager to hear your suggestions.
Regards,
Bhaskar

hi bhaskardutta,

what defines your positive control?
is it from some kind of culture.....?
did you know the concentration and purity of your DNA?
what is your PCR composition and profile?

Hadrian

Yes I did run PCR product in the gel too

My positive control is genomic DNA + plasmid DNA (1:1) from a E.coli culture.

The initial starting coc is 1mg/ml diluted serially for use as standard. 260/280 ratio is around 1.7


PCR is of standard composition with variations as mentioned in the above post.

Normally qPCR's primers are specifically designed for optimized reaction condition setup. In your case, I think maybe it's not a reasonable method to detect DNA, because different amplicons are randomly generated and no single amplicon molecule can accumulate specifically and reach the threshold of being detected.