Do a trial run with samples that are not precious.  Ask around for large weight  molecular DNA or just run the gel with a high molecular weight DNA ladder alone.  The one we used has been discontinued.  Space them out  evenly in different lanes across the gel.  Run the gel an see what happens.  I had to tweak the running conditions on a CHEF, before getting the kind of separation I was looking for.

Hi Amos,

I am currently having a similar issue like you had last year. How did you figure it out? Did you try running your samples with the bent electrode? One of my electrodes is actually broken and obviously disconnected at one end, though current still flows from same electrode. I have ran my samples without any result, and just wondering if that could be the reason for not getting results.....Could you share your experience and findings with me. Thank you

Hi Olakunle,

I was lucky.  I noticed the bend only after I had started running the gel so decided to continue running it. I then waited until the image was processed and saw that it looked fine. I guess that one electrode being bent isn't a huge issue (providing bubbles come out of the electrode). Because I was pressed for time I ran 2-3 more gels with the bent electrode before I could get it fixed and they were all fine. It was pretty easy to fix it but it happened on a weekend so we couldn't order new electrodes from Bio-Rad for a couple days. What I figured out is that the electrode is electronically connected only on one side. On the other it is just glued to keep straight. If you're lucky then it's the glue side that is broken and current still flows - like you mention you have. If that side breaks it isn't terrible. When you say you did not get results what do you mean?  No bands at all? slanted bands? My guess is that if you didn't see any bands it is not because of the electrode - if the electrode was the issue you'd probably see slanted bands or some kind of shift. Terry's answer above is a good way to check. 

Keep me posted. I would be interested to hear how things go for you.

Good luck,

Amos

Hi Amos,

Thanks for sharing your experience with me. It was really helpful to know about the intricacy of how the electrodes are connected. Yes, I didn't see bands at all, and it was really quite frustrating - I also thought as much that the broken electrode couldn't have been the issue, as in the worse case of scenario, I get a bent migratory pattern of my DNA on my gel....but that wasn't the case - no bands at all!. Another thought that came to mind was the orientation of my gel within the box. There is no indication as to where the well side of the gel should face - OR am I mistaken? Do you have any idea about  the position that my  gel should face within the hexagonal electric field? I have tried positioning my gel with the well side facing both left and right directions of the field, but still with no bands in either situations. I noticed in both situations that it appeared as though my DNA didn't migrate out of the wells. Should they be facing upwards or downwards - those are the other two options I haven't tried? I used 0.5X TBE (Tris base, Boric acid and EDTA at pH 8.3......or did you adjust your total stock solution to pH 8.3?), 1% Agarose, 20hrs runtime etc...and my current was ~157mA. 

I will appreciate your response to my concerns, as much as you can.

Appreciated and hope to hear from you soon. Thank you.

Hi Olakunle,

Yes, they wells should be on the upper part where the flow of buffer comes in. The DNA migrates in the same direction as the buffer flow. Here is the link to the manual Bio-rad have online:

http://www.bio-rad.com/webroot/web/pdf/lsr/literature/M1703690.pdf

It has a lot of good information about setting up the machine and troubleshooting problems. 

As for the specific run setting that will depend on what you need to achieve.  I used 3v/cm, 19hours and 0.5 s initial switch time and 7 s final switch time. 

Hope this helps,

Amos