I am following this paper (DOI: 10.1128/AEM.02895-10) to run a PCR that could differentiate between live and dead bacteria.
The method is based on propidium monoazide (PMA), which enters the dead cells, binds to the DNA, and then polymerizes, impairing the subsequent PCR step. The s s done with UV irradiation. The authors report this step as: "10 min of incubation in the dark, samples were exposed for 5 min to a 600-W halogen light source at a distance of 15 to 20 cm from the light source".
I have available a cross-linker with an emission at 254 nm but with only 5 W, not 600 W.
I have killed the bacteria by incubating them at 99 degrees for 15 mins. I added PMA at the suggested concentration of 100 μM in the dark for 10 mins to both the treated cells and a control. To compensate, I left the samples for 30 mins under my lamp in a tissue culture plate (to be sure that light reached the samples).
However, the PCR showed only a decrease of one circle in the treated samples compared to the control.
Am I correct in thinking that 5 W is too little? Or is there another fundamental step I am overlooking?
A 600 W lamp is not readily available. I have a 40 W lamp in the cabinet. Would that be better? and how long should the samples be irradiated?
Would the normal 1.5 mL tubes be transparent to the 254 nm light to are there special tubes?
Or is the fully chemical approach to differentiate live from dead bacteria by PCR?
Thank you
The lower wattage just means you need to extend the exposure time or reduce the distance proportionately. Yes a 40W lamp would be better. The petri dish/tissue culture plate approach would be best as you do not want any materials between your lamp and the cells. You also want as large a surface area exposed as possible to catch the photons. Consider using mirrors to focus the light onto the samples.
Following on this, I used the germicidal 40 W UV C lamp in a cabinet, for 15 min at a distance of about 5-10 cm. I got a 5 cycle reduction over the control (cells not killed by heat). Is there a way to improve the protocol? Does the exact wavelength of the light influence the outcome? In the literature there is a wide variety of values (365-460 nm). Thanks
If heat is an issue you might be able to use ice packs to keep the dishes cooler while you expose them. Yes the wavelength would affect the efficacy of the chemical reaction. If your lamp only produces frequencies outside the range at which the chemical reaction occurs you will not get the desired outcome. Try and look for a paper looking at the reaction rate for that compound against frequency (ACS might have one).
The heat is not a problem. But the germicidal lamps in the cabinet emit UV-C light (280 nm) instead of UV-A (400 nm). I am now trying to change the lamps. Thanks
Check the frequencies used by your gel docs/transilluminators. Older ones often use frequencies in the 360 range.
I used an UV-A lamp with a peak at 365 nm (Akynite Black Light Lamp UV E27 40 W, 3U CFL, UVA 365 nm), kept the samples for 30 min but there is no difference between treated (heated 15 min at 99 degrees) and controls (15 min at room temperature) on qPCR (not even 1 cycle difference).
I think one possibility is that the lamp is garbage and does not emit at 365 nm.
The other possibility is that the extraction ruined the PMA-structure. In particular, the kit I am using requires heating the samples at 65 degrees for 3 min; might that had affected the chemistry of PMA?
What else is left as optimization parameter?
Thank you
ADDENDUM: it turned out the problem here was with the extraction. After repeating, there is a 10 qPCR cycles difference between dead and alive bacteria, which is what I was looking for. Case Closed. Thank you for the support
ADDENDUM: actually, it looks like the reduction in copy numbers was just due to the UV exposure. If I mix alive or isopropanol-treated bacteria, the results are always the same, even with or without PMA.
What could be the problem for NOT having a reacting PMA?
Could it be that the PMA is already degraded even if the tube is just a couple of weeks old?
- Badges
- Science topic
- Similar topics
- Biological Science
- Microbiology
- Microorganisms