2 replies to this topic

[schookp]

Hello

I have the following problem and will take any advice I can get-

I am enumerating environmental samples in water through OD, plating, and qPCR.  My primers are previously published 16s bacterial targets (Maeda et al. 2003) and have great Efficiency and R^2 values with my lab-generated qPCR genomic standards (P. putida genomic DNA).  I am using the Mo Bio Ultraclean DNA extraction kit which results in a 50ul elutant with 5ul of that material used for qPCR analysis in a 25ul (total volume) reacion.

However, my qPCR results are not matching my OD and plating results.  In fact, they are lower than my plating results by 2-3 logs even when I use a lab-generated sample in dH2O. I have not been able to find much insight into how to correct this in the primary literature. I would greatly appreciate any insights that could be given for how to correct my qPCR results to match my plating counts.  I don't think its an extraction efficiency problem as both pristine and complex environmental samples are off by identical amounts, leading me to think its not an inhibitor in the suspension.

Do I have to multiply these values by something to correct for the amount of culture used, extracted volume, and volume added to reaction?  If so, should I apply the same calculations to my genomic standards generated by the same kit?

Again, any insights or advice would be most welcomed.

[jerryshelly1]

Are you trying to correlate your bacterial growth in liquid media and your transformation efficiency to your 16S RT-PCR results?

[phage434]

Low concentration samples often give problems when side wall binding to tubes becomes an important factor.  You might try siliconized tubes, or you could add irrelevant DNA to your samples to bind the tube walls.  I'd use something with a well defined sequence, such as a pUC19 sample. Low concentration samples of pUC19 are typically distributed with commercial competent cells.