DGGE gradient troubleshooting

[S.A.M.]

Anyone here does DGGE? I am having some problems getting tight bands in a certain gradient range and its driving me nuts.

I am using a DNA marker isolated from lab communities as a standard for optimising the gels. My initial gel run was 35-65 for 17 hours at 65V and 60 degrees Celcius. That was alright. I got a good resolution. Now I need to open out the gel between so I am doing 45-65 and 45-55 at the same voltage and time for consistency. Unfortunately, in the 45-55 range, the bands do not seem to be clear and the resolution is very poor, is it because of the narrow range? Its the first time I am using this gradient. I use 1XTAE as my buffer and APS and Temed as crosslinkers, I pour my own gels and use the Bio-Rad DGENE electrophoresis system.

The bands are very diffuse in the 45-55% and I have no idea why.

Any ideas?

 

[CharonZ]

Questions:
Do you mean that the bands are fuzzy and not clear?
The range has no effect on the fuzzyness of the gel. Did you run a marker on that gradient?
Essentially there are two main culprits for that kind of syptoms: bad PCR or bad gel.

What is the acrylamide to bis-acrylamide ration you use? Are all chemicals reasonably fresh (I assume so, as the other gels look fine, but then I don't know whether you poured them all at the same time).
How long did you let them polymerize?

 

[S.A.M.]

Questions:
Do you mean that the bands are fuzzy and not clear?

Yup the bands are diffuse and not as sharp. Since this is the marker, its a problem. I tried a quick 5h run at 200 V, same it affects only the 45-55

The range has no effect on the fuzzyness of the gel. Did you run a marker on that gradient?

I'm presently optimising the gel only with marker. I make the marker in bulk and aliquot it. Based on an optimisation for well volume [from 5 to 50 ul] I use 20 ul of the marker per well.

Essentially there are two main culprits for that kind of syptoms: bad PCR or bad gel.

The PCR should not be a problem, since I use the same marker across gradients

What is the acrylamide to bis-acrylamide ration you use? Are all chemicals reasonably fresh (I assume so, as the other gels look fine, but then I don't know whether you poured them all at the same time).

I use 40% bis:acrylamide and 8M urea. Since I am optimising I make the gradient with different volumes of 0% and 100% individually for each gel. Once its optimised I make bulk 35%, 65% etc.

I prepare the gel the evening before or morning of the day, depending on whether I am running an overnight gel or a 5h run.

How long did you let them polymerize?

I polymerise the acrylamide gel for one hour and the stacking gel for 30 minutes.

 

[S.A.M.]

One more point. I use a 15 band marker, in the 45-55 many bands remain at the top and many apparently go off the gel. So I get only a few bands distributed. Do I need a new marker for this gradient or will it affect cross comparison with the other gradients?

Also one of the primers I use for the PCR has GC clamps on it. Could this affect the resolution at certain gradients?

 

[CharonZ]

Sounds pretty basic to me. My best guess would be the gel itself.

However 1h is very short. Depending on minute pipetting errors, chemical quality and even changes in room humidity and temperature the polymerization may not be complete at that point. 1.5 - 2 hours (for the complete gel) are a much safer bet. Even longer, is possible unless the room air is very dry (but you can wrap the gel to avoid drying).
Edit: I did not read it correctly the first time. You said you prepare the gel the day before? So does it polymerize o/n. Or did you meant you just poured everything together sans APS and TEMED (or denaturants)?


Another thought: I am not familiar with the bio-rad system, does it run only one gel or several gels? In the latter case, was the bad gel positioned at the same as the others? Also, were the Ampere values comparable?

Regarding markers: you should give some thought which final markers you really need for your application. If you need to compare different gradients directly for some reasons, you need to use the same marker, or rather fragments with the same sequence. You could, of course make some marker fragments on your own (e.g. by pcr of defined length and sequence) and add it to the mix to increase resolution in certain areas.

GC clamps should not really have an impact on resolution. The effects on the PCR will likely to be more severe.

 

[S.A.M.]

Okay, I wondered if it could be the polymerisation time and was more obvious in a smaller range. I will polymerise for 2 hours tomorrow and run it for overnight to see what happens.

The DGENE system runs two gels. I use a block at the back when I run one gel.

GC clamps should not really have an impact on resolution. The effects on the PCR will likely to be more severe.

I wondered about that too, but I'm completing someone else's work and I'm not sure how much variability I can safely introduce.

Thanks for the input. I would put up gel pics but I'm not certain its ethical.

 

[S.A.M.]

Edit: I did not read it correctly the first time. You said you prepare the gel the day before? So does it polymerize o/n. Or did you meant you just poured everything together sans APS and TEMED (or denaturants)?

No, I prepare it in the evening for an overnight gel and in the morning for a day run. I add the APS and TEMED to the denaturants just before adding them to the gradient pourer.

 

[S.A.M.]

Update:

Just wanted to say thanks; I polymerised the gel overnight and it worked like a dream.

 

[CharonZ]

:=)

 

[S.A.M.]

I'm still wondering why the effect was so pronounced in the 45-55% as compared to the 35-65%.

Any ideas?

Also, on o/n polymerisation, the wells get a little dry, even though I wash them out with TAE and cover them with soaked Kimwipes and Saran wrap. Our lab is too dry maybe. Is there anything I should watch out for that could affect the gel quality?

 

[CharonZ]

To the first part I would guess that it was just chance. Slight difference in APS/TEMED concentrations, or inequal mixing or whatever. As the polymerization time was very short it was just not enough in this particular case. Regarding dryness, I had the same problem, I finally put the whole caster in to a tray filled with water and wrapped everything again into a plastic bag.
There are also a number of further things that may effect gel quality, though it tends to be somewhat less critical for DNA in general than, e.g. a 2D gel. One could, for instance degas the solution before pouring to reduce inequal polymerization. Or (depending on the gel system) optimize the running temperature. Depending on how much heat you generate and how warm your lab is, for instance.

But it really depends on whether you really need to increase the quality.

 

[S.A.M.]

Regarding dryness, I had the same problem, I finally put the whole caster in to a tray filled with water and wrapped everything again into a plastic bag.

Do you keep it out or at 4 degrees Celcius?

How long is a cast gel good for? Can I pour a gel on Friday and use it on Monday? I run 6 gels at a time and now I am running "day gels" ie for 5h at 200V, I usually go out of town on weekends, and I hate to lose one day because of not being able to come in Sunday to pour the gels.

I make 2 ml aliquots of the 10% APS [in freezer] and TEMED, to avoid differences due to exposure or preparation. I generally make enough for a full set [ie all the 35-65% for example].

I ran another 4 gels today and they turned out pretty well. Will be running all of one project next week, hopefully in time to send an abstract for EB. :yay:

 

[CharonZ]

The polymerization should be done at RT. After that you can store it at 4°C (wrapped). Depending on your fridge and how good the wrapping is, one can use gels a week or so. Possibly longer.

APS should be used up very quickly. Avoid storing longer than a week or so. What I meant however, was that as TEMED and APS volumes are usually low compared to the rest, there might be fluctuations in final concentration and thus in polymerization times.

EB?

 

[S.A.M.]

Experimental Biology, the abstract is due November.

I'll try one refrigerated gel for an overnight, sometime this week.