Hello everyone! I've noticed my DGGE gels have been having issues spreading down the gel and seems constricted to the upper half of the gel. In previous gels the marker is further spread out and I'm not sure why there is a change now.
I run the gels at 65°c in 0.5TAE buffer for 960 minutes at 60v in a CBS scientific tank.
I attached an image. I know it could be worse, but I've also made better and they've been like this consistently for awhile now.
Dear Medina,
I would suggest to ensure the gel casting step, I had a similar problem and i went to decrease the denaturing range, but after-that I realized that the problem was in the casting step. I was using biorad casting wheel.
So, maybe you try to reassure the gradient making is right.
Regards
Dear Medina,
I would suggest to ensure the gel casting step, I had a similar problem and i went to decrease the denaturing range, but after-that I realized that the problem was in the casting step. I was using biorad casting wheel.
So, maybe you try to reassure the gradient making is right.
Regards
For sure it is just a matter of casting step. Both casting wheel and gradient former (http://www.bio-rad.com/LifeScience/pdf/Bulletin_4006192A.pdf) can deliver pretty good and linear denaturing gel, independently from the gradient that you use. I would suggest to:
1. slow down the delivery speed of the acrylamide solutions (don't worry about fast polymerization, you have at least 5 minutes more than you may think, following the literature).
2. carefully mix temed and APS before delivering the gel.
3. check the tubing, use new ones or deeply clean the old ones.
4. check the glasses and the gel caster, since the spacer and the bottom gum must be in perfect conditions. You may also try to use paraffine oil on the spacers and bottom, to be sure that no leaking will occur. I don't like this second approach but sometimes is the only way to improve the gel quality
Hi Medina,
what is your gradient on that gel?
I'm using Biorad tank, with 100V, 60°C and 23 h running time (no joke) and after ~16 hrs it was not fully spread out. Sometimes if you are running two gels at a time, one is not spreading as fast as the other. I suggest run only one at a time!
Hi Janina, I'm using an 8% resolving gel, but I have been since January and it has been working well. I have been using the CBS scientific and I run 4 gels at a time and they ussually come out similar, and running one at a time isn't an option.
Mohamed and Luciano I use a peristaltic pump to pour the gels with a gradiet maker (pour low and high and there is a spinner at the bottom to mix them as they meet and go out into the pump). I can't adjust the speed on the pump, there is just forward, off, and backwards as settings. Do you think it could be caused by the spinner not working properly and thus the high and low acrylimide colutions are not mixing properly?
Hi Medina,
8% is your acrylamide content but what is the gradient of the denaturing agents? Like 20 to 70% or 40 to 70%? What is the size of your PCR fragment?
sorry! It is 40% to 60% and the PCR fragment is about 370 bp. it is the V2-V3 region of the 16s rRNA
Hi Medina, me too I currently use a peristaltic pump without speed control. you can put a rotating magnet just in the high solution chamber. remember to check the tubing wit water before pouring acrylamide, I do this all the times, before casting. Also, check all your reagents, if Acrylamide, formamide, ammonium persulphate and TEMED are all before their expiry date. I had once problems like yours just for the APS. Don't store too much the acrylamide solution, I would suggest less than 15 days.
Hi Everyone,
I spoke to my supervisor again today and he's having me try a new recepie with 16ul temed, no glycerol, and leaving space for a 3.5% stacking gel. I shall have answers by Thursday if this has fixed the issue. Thank you for your input, and the next step should the new recepie not would would be ordering a new bottle of Formamide!
Hi Medina, I doubt you have a problem with old reagents, your bands would be more fuzzy then. Is the middle lane and lane on the right side your marker? Is the spread unusual for this marker? You could still have a community of low G/C content...i would try higher voltage and running times!
the first, middle, and last lanes are my markers. The one of the left has a lot of smiling, so when I figure this out I will run less at a time so that won't happen. Usually they are more spread out, as seen in the attached pictures. I'll see what results I get tomorrow and if they're still squished I'll try the longer running time!
Hi Medina, these gels are nicely spread out!
The smiling effect has a different reason. It's because urea and formamide leak out at the edges forming another small gradient. There is a simple trick, just use grease between spacers and glass plates when casting the gel as described in this paper.
BRINKHOFF, T., & HANNEN, E. J. (2001). Use of silicone grease to avoid “smiling effect” in denaturing gradient gel electrophoresis. Journal of Rapid Methods & Automation in Microbiology, 9(4), 259-261.
This was todays gel with the new formula. While it does look better, it still isn't as spread out as my boss would like. We are now doing a 6% acrylimide gel, and then we are going to try reducing the amount of formamide by 1/4. Any other input would be appreciated!
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