I have been trying to use the reverse line blotting to detect several tick borne pathogens. However, I get very large signals. I have decreased the concentration of both PCR products and the probes by half and still have the same problem. The hybridization temperature I use is 60C.
Using Millipore* Immobilon* Western Chemiluminescent HRP Substrate (ECL) then I expose the membrane to Fluor-S Max multi-Imager. I changed the exposure time from 180 sec to 50 sec with the same signal size.
Do you by chance have an example image?
We use the new version (just came out) of the Biorad Chemidoc and we expose for 1 minute to get the general overview and adjust the exposure time accordingly.
With the 1 minute exposure, it is possible to see the weaker signals and if there are results that are too intense, we can then adjust the exposure time. (Sometimes even down to 20 seconds or less) The two images will then give the complete result of the blot.
As I am not familiar with the Fluor-S Max multi-Imager, I do not know if it has this feature but we can also select certain regions on the blot which the imager takes as reference points. The imager will then use these parts of the blot in an automated exposure until the signals in the selected part of the blot are reaching the set threshold. This option does, however, have the risk of not detecting the weaker signals so therefore we always go for a 1 minute exposure to start with.
But just to ask, what is exactly the dilution of your PCR product and what is the used probe concentration that is bound to the membrane? And did you develop the RLB setup yourself or was this published before?
The pictures format is not compatible with the researchgate. The PCR concentration I used was 20ul PCR product/ 200ul Buffer and then I reduced it to 10 per 200 and still have the same large signals. For the probes, I used 5Picomol then reduced it to 2 and still no change. I am following this paper http://www.nature.com/nprot/journal/v1/n6/full/nprot.2006.404.html
Ahh not a problem, i am happy it works now.
About the probe concentration, we usually use 2,5 pmol/µl and adjust this after evaluating the first blot (according to standard controls). Sometimes probes are not that efficient and it is better to add a bit more to receive a better signal.
PCR product wise, we use 10µl per 160µl total volume, but we do not use a multiplex PCR. We run separate PCRs per genus, which we then combine into the 160µl total volume that will be loaded onto the blotter. (usually 4 x 10µl in a total volume of 160µl.)
You can contact me if you have any further questions by the way.
Which polymerase do you use? does it have to be a hot-start one?
It does not have to be a hot-start one, but in that case i would recommend to setup the pcr reactions on ice to prevent false annealing.
We use Phire, which is a hot-start polymerase, in a lower then recommended concentration. 0.125microliters per 25microliter total pcr reaction volume. (Recommended would be 0,5microliters per 25total volume according to the manual, but then the signals are very strong and sensitivity testing has proven that 0,125 was sufficient. This also makes it very cost efficient.) But as this polymerase has proofreading, you can not use a uracil/udg step to prevent old amplicon contamination.
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