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I amplified my DNA with real time PCR. The amplification curve shows good amplification but not a single band is observed in my gel after gel electrophoresis. What would be the possible reasons?

I am attaching a photograph of my gel below. My ladder opened correctly but not a single band can be observed:

                                          Gel

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  • $\begingroup$ In which cycle did your sample come up? Are you sure it is your wanted product and not a primer dimer (which are visible on the gel)? What is you expected product size for your PCR product? How much DNA from your qPCR did you load on the gel? $\endgroup$
    – Chris ♦
    Nov 10, 2014 at 20:11
  • $\begingroup$ almost at 30th cycle.yes these are primer dimers. i load about 5ul of pcr products in my gel. $\endgroup$
    – tanzeela
    Nov 10, 2014 at 20:17
  • $\begingroup$ my product size is 170 base pair $\endgroup$
    – tanzeela
    Nov 10, 2014 at 20:18
  • $\begingroup$ Do you know that you have product besides the primer dimers? How does the melting curve look like? There you should see two melting peaks - one for your product and one for the primer dimers. Besides that, 30 is relatively late, so there is not much template. You could of course precipite the whole PCR reaction and then concentrate it to see the product. $\endgroup$
    – Chris ♦
    Nov 10, 2014 at 20:22
  • $\begingroup$ yes there are two melting peaks.but i quantify my pcr products with nanodrop method; quantification shows that signifacant amount of amplified dna is present $\endgroup$
    – tanzeela
    Nov 10, 2014 at 20:27

1 Answer 1

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As you can see from the bottom of the gel, the brightness of the bands have reduced even for the DNA ladder/marker. The main reason for this is that when you are running an agarose gel containing EtBr, due to the positive charge of the EtBr, the stain runs to the negative chamber, against the direction of the DNA migration as @Chris has pointed out! Although some of the DNA will stain but the rest of the EtBr will migrate towards the top of the gel (where the DNA is loaded).

A quick remedy for this is once you ran your gel is to soak your gel in a separate container containing your stain and running buffer such as TAE. I do this for one hour and that makes a huge difference!! Also make sure you regularly change the buffer in your agarose gel running apparatus and don't use the same buffer for a long time since again as @Chris has correctly pointed out there is a reduced conductivity in the older buffers when they heat up so migration does not work as well! Obviously this response assumes that you have a good degree (amount) of DNA in your gel bands, which can be calculated by nanodrop once you extracted the DNA using an extraction kit, or whatever method you normally use. I hope this helps!

This answer is more suited to the title of the question since after reading the main question a bit more carefully I'm slightly confused! So do you see anything at all or your product is far too faint? could you tell us where on the gel, relative to the ladder you expect to see this band? are they the ones at the button of the gel, below the loading buffer?

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  • $\begingroup$ Your assumption about the Ethidiumbromide is wrong. In solution it is charged positive so it runs to the kathode and against the running direction of the DNA. Some of the EtBr will intercalate into the double helix and stain it (although weaker the further the DNA migrates), the rest migrates as long as the power is on. This is why it is usually recommended to stain gels which run very long afterwards, as this increases the staining. $\endgroup$
    – Chris ♦
    Nov 10, 2014 at 21:55
  • $\begingroup$ @Chris Ah, but I was told it is the pH effect that causes this since in my apparatus containing old running buffer this is worse than when made freshly and when I use the buffer in the positive chamber side of the apparatus and sock in EtBr, it doesn't stain. But I could be wrong! I can edit my response if thats best? $\endgroup$
    – Behzad Rowshanravan
    Nov 10, 2014 at 21:58
  • $\begingroup$ No, the pH has almost no effect on this. You will loose conductivity in older buffers as well when it heats up, so the migration is not working that good anymore. You could add EtBr also to the lower buffer reservoir, but this can get messy. $\endgroup$
    – Chris ♦
    Nov 10, 2014 at 22:04
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    $\begingroup$ @Chris ah interesting! thanks! I shall edit my response! $\endgroup$
    – Behzad Rowshanravan
    Nov 10, 2014 at 22:05

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