DGGE Migration Issues - (Sep/22/2014 )

Hey folks,

I'm running a DGGE on faecal and clonal DNA samples (V3 segments of bacterial 16S gene), and while the results are roughly as expected band-wise, there's a little problem with the gel; namely, the band patterns seem to start about halfway down the gel instead of at the wells as expected. You can see what I mean in this picture:

(Ignore the actual bands for now, that problem's already been resolved)

Some background: I haven't been using a gradient former, instead using this method to pour the gradient gel. Similarly, since our lab doesn't have a proper DGGE system, I've been using a Bio-Rad Mini-Protean Tetra System in a water bath to keep it and the buffer at 60 'C. I'm not sure if this is relevant or not, since the DGGE seems to have more or less run properly, just not in the upper portion of the gel.

I'm sure this isn't just a case of all the bands migrating to the lower half of the gradient, as I've tried a few more gels with different gradients and all of them are producing something similar - a smaller gradient of 40 - 60% actually looked like the top part of the band was being cut off at the halfway point. Also of concern is the presence of the 'dividing line' that marks the start of the gradient being present even in lanes where no sample was loaded.

Anyone else experienced this or can offer an explanation?

i haven't experienced this (mainly because i don't use that haphazard method of pouring a gradient and don't run dgge) but, it looks like you have an extended section of low %age gel (that the gradient didn't form all the way up).

we require more information to make more than an educated guess.

(are you really running a 40-60% gradient?)

do you add loading buffer to the unused wells? if so, then that can account for the line in the sample-free wells (so can the electrode buffer).

That gel there is a 30 - 65% gradient, and another ran with 40 - 60% produced the same halfway gradient start, but with the faecal sample band pattern looking like it had been cut off. I did this because I initially assumed the same thing you did - that the gradient pouring had gone a bit funny and left a large section of low % gel - and reckoned increasing the % of the lower boundary of the gradient might alleviate the problem somewhat.

I also thought that, due to not having a proper heating element, fluctuations in temperature might have been melting the upper portion of the gel (due to heat generated from passing high voltages through the system). However, upon running another gel and monitoring the temperature carefully, this wasn't the case - temperature was maintained exactly at 60 'C.

No loading buffer (or anything else) was added to the unused wells.

If you need any more specifics, please let me know - this is my first time running DGGE, and it's quite possible I'll miss relevant things!

i'm confused. page of nucleic acids is performed in anywhere from 4 to 8% acrylamide (depending on the size). one paper that i looked at, where dgge was used, was using 8% gels.

if you haven't used loading buffer in the empty wells then the line is from something in the electrode buffer.

mdfenko on Wed Sep 24 14:01:07 2014 said:

i'm confused. page of nucleic acids is performed in anywhere from 4 to 8% acrylamide (depending on the size). one paper that i looked at, where dgge was used, was using 8% gels.

My apologies, that's my fault for being unclear - the 40 - 60% gradient refers to the denaturant concentration (where 100% = 7M urea and 40% formamide). The acrylamide concentration is constant at 9%.

the gradient that is demonstrated in the video that you linked is an acrylamide gradient. gradient formation of additives won't necessarily work as well.

That could well be the root of the problem - I'll ask my PI if the lab can invest in a proper gradient former. Thanks for your help, I'll report back if results improve.