DGGE - samples not moving in well!! - (Apr/21/2004 )
I was hoping that someone might have some insight into a problem I have recently experienced with my DGGE's. Normally, I use a 35-60% gradient and a 45-55% gradient when running a parallel DGGE. Recently, because I am using new primers, I started using a 10-90 % gradient and a 25-75% gradient. Everything if fine until I come back in the lab 16 hours later to find that my samples have not migrated at all. This has happened 2 times so far this week. In fact, since I changed my gradient, I have not had a successful DGGE. ??? It doesnt make since that this new gradient would prevent my samples from moving? My new primers are 907-341 (GC), and I adjusted the acrylamide appropriately for the size of the new product. So, that shouldnt be a problem. And no, I am not plugging the cords in backwards! The power source works just fine when running agarose gels. I dont know--does anyone have a clue?
The 10-90% gradient is offering way too much electrical resistance(very low current). When you start the run (overnight I assume), what are your power, voltage and current values and what are those values when you come back after 16 hours? With this information, we can diagnose the problem. Most probably you may have to quit the parallel runs. Maybe you have to use TAE instead of TBE buffer. Maybe you should use 1.5X TAE instead of 1X TAE. Maybe you should check the acetic acid used to make the TAE or the pH of TAE used. Possibilities to troubleshoot are endless but we have to start with the electrical parameters at start and at finish.
If you have Bio-Rad's DCode sysytem to run your DGGE gels, you could have picked up the loading cover, then put it on backwards, causing the power supply to not deliver a current. One power supply gives us an error code when we do this, while the other power supply we have does not give an error - good luck.