Hello
I am running a PCR to quantify bacteria (namely Escherichia coli). I would like to count only live cells and I have seen there are several protocols that employe chemical dying of the death cell's DNA followed by DNA purification and then qPCR.
To avoid the costs of purification, is there a protocol for differentiating live from dead cells that includes simple boiling of the cells followed by PCR?
Thank you
Assuming the dead cells are porous or at least prone to pore forming agents you could do a DNAse digest first.
I don't think the question itself makes sense. What does live bacteria mean? Living? If you are going to do a PCR, it does not matter if the bacteria is living of dead because you are using DNA for PCR.
And if you asking about skipping the DNA extraction, then you should have read something about colony PCR.
Also, if you mean live with respect to visual aspect, you can easily use dyes for the live dead cell staining but that would not proceed further to PCR.
All in all, the question does not make sense and there are some vital information regarding molecular analysis which the questioner need to attain.
By 'living' I mean: quantify the DNA of bacteria that are still metabolically active as to reproduce the colony counting procedure, which is based on enumerating metabolically active (live) cells. A microscopic examination will not give me a precise quantitative answer either. A colony PCR requires making a plate, which I want to avoid. Brinzer's answer goes straight to my point.
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