All’s well that ends well–the aftermath

So I ran the last little bits of the samples that were ruined by the infamous E 9 message and they turned out perfectly, all samples amplified.  You can see in the picture the only drawback is that they are a bit faint, but for 3 uL loaded, I’m pretty impressed.  Yay!  At this point, there is only one more 96 sample PCR reaction left before all samples have been genotyped for coq-211 microsatellite.  We are expecting some new equipment for running gels today, so hopefully I will be able to run through the next microsatellite more quickly.

E 9: What it is and Why it’s BAD.

There are several things to watch out for when running gels of any particular sort, and this is especially true for DNA-PAGE.  Or so it would seem…

Error messages coming from the equipment are never a good thing.  There are a few that I’m already familiar with (unfortunately).  One example is Error 6, coded E 6 on the BioRad PowerPac 300 (note:  add lightening bolt between Power and Pac).  This error message means that the machine has shorted out or there isn’t enough buffer in the chamber to allow a current to pass through the gel and move the product as is the point of running a gel.  This message is absolutely infuriating when you’ve started a run to go overnight and you come in bright and early and find that sometime during the night (and you have no idea when) the gel has stopped running.  What do you do?  Run the gel a few more hours and risk running the bands off the gel?  Or hope that an hour is enough and then take it out and stain it?  The answer is avoid this error in the first place, of course.  Running 4 gels at a time tends to decrease leaking of buffer, which is the most common reason for this error message.  But if you have not choice and you’ve taken every precaution and it’s just gremlins out to get you, then go ahead and run the gel for a few more hours.  Chances are, the gel stopped running less than an hour in.

But on to the point of this post:  Why E 9 is BAD.  Well, if it happens, this is what at least 2 of your gels will look like:

And that’s after a 5 hour run.

So what happened?  Error 9 means that a connection has failed in the gel apparatus somewhere and the current, while running fine in the other 2 gels not attached to this connection, isn’t moving the PCR product along like it should.  BioRad instructions say that all you have to do is turn the machine off and than back on again to fix the problem.  So that’s what I did.  Clearly, this is not the best method.  Unfortunately, I don’t have a fix-it for this one either.  But, what I can say is that it is always good practice to leave a little PCR product behind in the tubes when loading, so if something crazy like this happens, you don’t necessarily have to do PCR again.  Which is something of a relief if, as you can plainly see in the picture, the reaction worked.  A small success.