Well, approaching this problem sequentially:

-> If the DNA is the same as in successful gels, either the DNA suffered severe degradation (heat-exposure and/or DNase contamination), or DNA is not the problem.

-> By the looks of the gel, DNA is not the problem; you have adequate size and amounts of DNA, it is just not stoping in the appropriate place; ergo, there's something really wrong with the DGGE itself.

-> In DGGE, several thing can go wrong: the gel, the electric run, the temperature and the buffer .

a) Gel not properly made - gradient is all messed up.

b) Temperature is above 60ºC and/or not constant - the apparatus is broken or you are using 2 gels per run (which is not a good idea -> it will increase resistance (mA) and normally the 2nd gel "back gel" gets too close to the heating part of the apparatus).

c) Buffer is old - from my experience, you shouldnt re-use the buffer, but if you do, maximum 3/4 runs per buffer batch...

Does any of these sugestions help or indicate a probable source of error in the gels you showed?

Hopefully helpful,

Rafael

Hi Rafael,

Thanks for your help. In reply to your suggestions, in order...

a) I think this is very possibly the problem. I'm using the Bio-Rad gradient former, where you just turn a wheel and it pushes the syringes of high and low solutions. I have quite a steady hand but this is not a very good method of pouring a gel! However it did the job fine when it worked the first 3 times. I'm not sure what else I could try.

b) I actually set the temperature initially to 63ºC, load the samples, and by the time they are loaded and I put the lid back on, it's cooled to 60ºC. If not, I wait until it is exactly 60ºC before I turn on the power pack. Do you think that would cause a problem? I only use the "front" gel. I just have empty glass plates in the back one.

c) For most of the above pictures, I used new buffer. But if not, I tend to re-use it about 3 times max. 

Thanks again,

Lindsay

I think 63ºC would be fine, and never the cause for this type of distortion... If your are not using the back gel, then thats fine too. The buffer should be ok...

That leaves us with the gel itself...

Just to know if the gradient-maker is the problem... you should first rule out other possible weak-points:

-> Is the buffer used to make the solutions fresh?

-> Are you preparing the Weak and Strong solutions fresh, or are they prepared in excess and frozen for subsequent uses?

-> Are you preparing 10% APS fresh? This one is critical.

The buffer is made fresh, yeah.

The high and low solutions...I don't make them fresh every time. One of those pictures above will have been made with a new solution, but generally I store it in the fridge at 4ºC for up to a month (I think this is what it recommends in the user manual?)

I use fresh APS every time.

Just in order to add some more considerations:

- what about the electrophoretical running conditions (how mwny Volts for how mwny hours)?

- are the starting materials to prepare the high and low solutions good? Where do you store usualyy acrylamide/bis?

As a general consideration BIORAD Gradient Maker is not the top but anyway it works, altough a peristaltic pump wold be better, but indeed looking at the pictures the problem would seem to be attributed to the starting materials (that could influence a good polimerization) or to the way the gradient is formed, moreover taking into account the fact that you run a front gel (because very often the back gel suffers somehow in the BIORAD system).

Im not sure this could really help you but I hope so

Maurizio

Hi Maurizio,

I run at 120V and for the above images, the times were 12, 11.5, 10.5 and 9 hours. I was trying different times to see which would work. 

I store the stock acrylamide/bis in the fridge at 4ºC. Would you recommend making fresh high and low density solutions fresh before each run? Currently I make a bottle of each and store them in the fridge for up to a month. 

I also prepare a 250 ml bottle of 0 and 100% denaturing solutions and store them for up 1 month at 4°C, this cannot absolutely be the problem.

On the other side in my experience I usually run the gel for 16-18H overnight at 70-85 V and I am conscious that people who run the gel at a high voltage like yours ususally run for no more than 6 -8 hours; a too long running time at high voltage might explain at least the U shaped bands in your gels

Maurizio, you say that you store your 0 and 100% denaturing solutions - I don't ever make these. I use a 30-60% gradient and make up bottles of high (60%) and low (30%) denaturing solutions and keep these at 4°C. What do you use the 0% and 100% solutions for? As stocks? Do you think that would make a difference?

In terms of voltage, I thought 120 was normal, as I have seen people using 200V before! But I will try your suggestion and run at 80V next time. Do you think the buffer would make any difference to run time? I use 1X TAE but one of my supervisors used 0.5X at 80V for 17.5 hours - could I use this voltage and time with 1X TAE?

Thanks!

I do also start with a 0 and 100% denaturating solution as my stock and then I make my working solutions when I need them. I use a 30-60% gradient.

The voltage i use is 50V for 18h when i do a dgge. I also use 1× TAE so that should work fine. 

I also started at a voltage of 120V  for too long and I had the same problem like you. Lowering the voltage did help in my case.

That's great Pieter, thanks very much for your help. I will try 50V for 18h next time.

Do you use a 0% stacking gel to stick the comb in on top of your gel? Some kind person on here sent me their protocol but it's completely different to mine. I had just been using the gradient former to make the entire gel and sticking the comb in the top where the denaturant concentration would be 30%.

I always use a 0% stacking gel to stick the comb. In that gel the dna forms one good band before the denaturation starts. Otherwise the denaturation can already start when you are loading and it is possible to get some smiling then.

Great, I will definitely have to start doing that then! Thanks so much for your help!

If all the previous suggestions don't help, you might want to check your formamide.  Could it have been left somewhere warm in between setting up the gels that worked and those that didn't?  It's usually the component that goes off first, so I store mine in the freezer.

As an aside, what kind of gloves are you wearing for handling your gels? The fourth picture down has some smearing that looks like it might be from fingers.  Nitrile gloves tend to do this, so you might want to put latex gloves on over top if you use nitrile.

Good luck!

Alison

Thanks Alison, I keep my formamide in the fridge and only take it out to use it for a couple of minutes and put it straight back in. Nobody else really uses the fridge and I'm the only one in the building doing DGGE so I doubt anything could have happened to it.

I do use nitrile gloves! I noticed the fingerprints myself. I will try and get my hands on some latex ones. I was actually wondering if it makes a difference if you use the same tub for both staining and de-staining?

Has anyone had sucess using other stains other than ethidium bromide? It came with the kit and I didn't think it'd be worth buying something different (we use SybrSafe in our lab but I know it won't work for this).

I've never had a problem with using the same tub for both staining and destaining, but it might depend on the material the tub is made of.  I used glass for ethidium bromide. 

The SYBR Green I stain works well for DGGE gels, but it partially depends on how you are visualizing the gel.  It worked great with a Typhoon, but not with a standard UV box. If you have the correct filters then the UV box might be OK.

I use SYBR Gold to stain my gels. It is more sensetive then ethidium bromide and it works very good for me.

I prefer to do the de-staining in the same tub. I have seen that the less I have to touch te gel, the better that the pictures are.

One major problem could be the gradients of the gel you use itself. Improper gel polymerization leads to incomplete gradient formation which is known to cause patterns like these. Please check the acrylamide solutions and whether they are stored properly. One step you could do is not to store the gradient solutions for long even if under 4C temp or below 0. Try and prepare the gel solutions fresh and use them immediately. Repeated thawing of solutions to room temperatures sometimes inactivates the urea and formamide used in the solutions. Hope this helps. Cheers

We use silver staining method for DGGE gel staining. And it is working good.

Thanks so much Alison, Pieter, Harish and Priyanka. I'm going to make some fresh solutions just now and add the stacking gel on top, which I hadn't been doing previously. Fingers crossed it works this time!

Hi

I do not think you have any problem with staining. It looks like the gels are bad. Make fresh reagents. The formamide and gradient solutions go bad easily. The curvy bands in one of your gels are due to improper polymerization. How much APC are you adding? The running conditions for the 2nd gel in your pictures are providing you good resolution of bands.

Hey guys, so I tried adding the stacking gel and ran it at 50V for 18 hours. It still didn't work (pic attached). I guess it must be reagents.

Rishu - I add 144ul of APS to 16ml of denaturing solution. This seemed to be the correct amount according to the Bio-Rad instruction booklet, if my maths is right!

The sample on the left is that you ladder?? Cause the samples on the right don't look that bad. The bands that I can see look good but there aren't a lot of bands. Do you expect to see a lot of bands??

I make my stock solutions fresh every week.

To make 16ml of denaturating solution I use 73 µl APS and 14.5µl Temed

Yeah that's a ladder but it's just a standard ladder, not meant to be used for DGGE but I just wanted to see what it would look like. It gave distinct bands last time I used it. I was expecting a lot of bands yeah! See attached pics for the same samples in June.

I made my "stock" solutions as 0%, 30% and 60% denaturing solutions on Monday and used them on Tuesday, so they were fresh then. I was hoping that the problem with the other gels was the old solutions but obviously that wasn't it. Unless the problem is the fact that I make the high and low density solutions as stocks rather than the 0% and 100% ones? Would that make a difference?

I'll try adding half the APS next time also.

Thanks :)

I don't think it's a problem to make your stock solutions at 30% and 60%. I can' think of a reason why this could be a problem.

And are you sure that the concentration of your DNA is high enough? Cause it's strange that in June you have a lot of bands and now there are almoust none. I would expect that the bands are there now also.

Oh no, if the DNA is the problem then that is really an issue! The DNA is a confusing story because I went to check it was still ok after being extracted a few months ago and stored at -20. And this is what it looked like (see 1st picture - note every second well also contains sample (from different GI tract location) - although it doesn't look like it! My supervisor told me to PCR it anyway. I didn't think it would work but then see 2nd picture for resulting products. Between the last band and the ladder is -ve control. The last band is +ve control which is the product from pure genomic DNA from mixed species that I ordered specifically.

The collection of weird looking gels in my original post used a mixture of these new products and some old ones which definitely worked nicely in DGGE before. Also on each gel there is a +ve control, so I would have expected nice bands from at least two lanes in each gel.

The DNA looks fine! So that will not be the problem! 

I was just thinking, how much mA do you use when you run a gel?? I use 50 mA for one and 100 mA for two.

I have no idea about the mA - I thought you chose to control either the V or the mA and the machine adjusted the other accordingly? I didn't know you could control both! I'll check next time I run them. I've just bought new formamide and acrylamide/bis so fingers crossed for the next gel!

With the system I use I have to change the mA manually. But it could be that it is different in other systems. Good luck!!

Ok I will have a check of that. Many thanks for all your help; it's very much appreciated!

How many hours for a gel setting?

Normally the gel will be set after 1h (even faster). But I mostly make my gel in the morning and I load it before I go home so it can set for 5h. Then it's for sure fine! If I have to work fast, I leave the left overs of the gel in a tube to see if that does set. If the left overs did set you can be sure that the gel did set also.

Your problem might be a matter of using the wrong cleaning agent for cleaning your DGGE device and the GLASS PLATES.

When we first realized our problem we started to work on the PCR/ the gel/ the buffers/running conditions .... EVERYTHING ... however, it took us month to realize that the soap for cleaning our DGGE device caused such smear effects!

We had changed it without puztting much attention to it. In the new soap - there where some oily substances added that interfered somehow with the glass surface. We used new glass plates and switched back to our old cleaning agent (kind of scrub cleaner without any special ingredients). Ask the supplier of your DGGE device for the best cleaning agent to use!

Please is this thread still active?

I have a lot of questions to ask on PCR-DGGE. My project involves using this protocol. Please help

Olalekan Oladimeji Akadiri It had been a long time since I did the DGGE now, but I can try to help! You can send me a private message if you like.

@Lindsay Fulton please check your message box, I sent you a message as regards DGGE