I am hoping to perform some RT-PCR. However my bacteria has taken longer to reach the correct OD at which I should harvest. Does this reflect in anyway the RNA which I then prep and take forward to use for the RT-PCR? Previous growth curves have suggested within 27-30 hours is when I should reach optimal OD but my bacteria don't grow this uniformally all the time. Would it be best to scrap this material and start again?
Probably, it depends on bacteria. I work with Salmonellae which are very fast-growing bacteria, and from my experience I can say that if ever you have doubts about the normality of growth, stop the experiment right away. What you risk to have at the end is an uncertainty about the actual conditions of the experiment and the validity of comparisons. Naturally, when you analyse a gene expression you try to exclude all extra factors which are likely to influence interpretation. The growth stage is one of such factors.
Hi Alexander,
Yes that was the decision I came to anyway. It is frustrating but I don't want to compromise my experiment.
Thank you for your response.
I agree with both of you (Alexander and Simone). Also, I recommend you consider plating out your stock every time you grow more bacteria for RT-PCR. I suggest you use at least two different media, preferentially differential media and _not_ Luria-Delbrück or other media used for bacteria genetic studies.You may see morphologic changes in colonies that indicate that your stock has gone bad, when otherwise you may think that you mis-measured your growth medium or made another error.
I guess its always better to rely on OD which should be 0.6 ideally for optimum growth of the bacteria, obviously as you are isolating RNA, (I suppose mRNA), the expression of any gene severely co relates with its growth stage, as you have not mentione your bacteria, my suggestion is isolate the RNA may be a bit prior to the optimum growth..it wont affect the results ..do it once, see the results then think of a plan B.
Best of luck,
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