I ran PCR for the detection of M.tb infection in clinical specimens. Sometimes I get the PCR product as a faint line while running the gel. Could anyone tell me how could I resolve this?
TBE gels tend be better at resolving than TAE gels. As Veera mentioned, a higher percent gel (but somewhat thin gel) will also help in resolving. Is it possible for you to increase the reaction size of your PCR so that you have more sample to load on the gel? Or can you run your PCR for more cycles so that you have more product and thus a stronger band on the gel? Also, the sell high-resolution agarose which also helps if you aren't already using it. Sometimes you must let the agar soak in the buffer for several minutes before melting it into the gel for it to pour properly.
What is the size of the pcr product? if it is less then you should go for a higher percentage gel.
TBE gels tend be better at resolving than TAE gels. As Veera mentioned, a higher percent gel (but somewhat thin gel) will also help in resolving. Is it possible for you to increase the reaction size of your PCR so that you have more sample to load on the gel? Or can you run your PCR for more cycles so that you have more product and thus a stronger band on the gel? Also, the sell high-resolution agarose which also helps if you aren't already using it. Sometimes you must let the agar soak in the buffer for several minutes before melting it into the gel for it to pour properly.
A number of questions: Is your PCR working ok (do you have a bullet-proof positive control which is working)? How do you treat you samples, do you use raw material or do you purify it? How much of your PCR reaction do you load? Which size has your expected PCR product? What is the percentage of you gel?
How molecular length marker looks like on your gels? If normal, then the problem is likely to be found in PCR.
We also prefer to use TBE gels for electrophoresis. I suggest to you check your TBE buffer. If it is prepared since a long before, you will need to prepare the new one. Furthermore, agarose should be dissolved well in the buffer and you see the transparent mixture.
using TBE and lower voltage/longer electrophoresis times helps resolve bands better - you should also make sure you are adding enough PCR product, that the gel is the correct concentration for your product and that it has completely set before loading. You should also run the gel immediately after loading so that the DNA does not diffuse through the surrounding gel - same for taking an image or extracting band from gel as soon as possible after the run has finished.
If you have very little template in your PCR reaction, you will get very faint bands, maybe even undetectable.
Have you tried playing around with the exposure settings on the transilluminator?
How about your DNA quality? Unclear band will appear if DNA qualit is poor. Usually we use 5 ul of PCR product and 1 ul of ehtidium bromide. Longer electrophorensis time may produce the dark band. After loading, run start at 100 Volate about 30 min can result the clear band if the quality of your DNA or PCR product is enough.
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