We genome labelled one gram positive bacterium with GFP having zeocin as selection pressure. This labelled bacterium which is showing fluorescence even in absence of the antibiotics. After isolating DNA and performing PCR for checking presence of GFP at genome level, proper band obtained. We infected rice plants with this labelled microbe in an enclosed setup and tried to reisolate them by spreading on plates having zeocin. Where control sprade-plates (having extract of untreated rice) show no colony in presence of zeocin but experiment sprade-plates containing zeocin (having extract of labelled microbe treated rice) show a few colonies. But no fluorescence seen and no proper band found when we did PCR using the same set of primers(what we previously used for checking the presence of GFP in labelled microbe) from DNA of isolated microbes from the infected rice. Why is it so? That same bacterium (wild type) can not grow in presence of zeocin. Will be glad if you kindly help me in this.
Regads
Trina Roychoudhury
Just a few quick questions. Were the GFP and zeocin genes part of a plasmid or stably integrated into the bacterial genome?
Did the positive control work for your PCR check of your isolated (post inoculation) colonies?
Amy Klocko Yes, GFP and zeocin are part of a plasmid which is mini-Tn7 vector. It should be genome integrated as per the protocol. No band was found when PCR was performed using DNA of isolated microbes(post inoculation) as template. So I did re-PCR using the first PCR product as template (1:10 diltion) and same set of primers were used. NO band, only smear obtained.
Ah, no bands at all sounds like a potential PCR issue. Hmm, do you have a sample of the plasmid to use as a positive control? You'll need to dilute it quite a bit. I usually do 1:100 dilution of a miniprep and use 1 ul of the diluted sample as a positive control. I can be challenging to do a nice clean PCR of a PCR, especially if it has been stored for a while. If there was any standard loading dye present that will inhibit your polymerase too.
Amy Klocko Thanks for your advice. I did not perform PCR from the plasmid. But I think it is highly unlikely not to get a band from PCR of the plasmid as I already got the band when did PCR from the DNA of GFP-labelled microbe (pre-inoculation one). I also think that PCR of a PCR is causing the trouble. I did PCR of a PCR using 1 day old PCR product stored at minus 20 degree C. I also tried using higher amount (2.5 times of 1:10 dilution) of template for PCR of a PCR. Again smear appeared but no band found. Using Phusion High- Fidelity DNA Polymerase (Thermo Scientific) for this purpose and no loading dye was mixed with the PCR product. This time, I will do the first PCR and will do the second PCR on the same day (1:100 dilution and will use 1ul of the diluted PCR product as the template). Hoping to get the band. Will update my result. Appreciate your help.
Sincerely,
Trina
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