Hello all,
I am working on a restriction digest of some DNA samples and I keep getting these dots/flame looking pattern that I can't seem to figure out. Can anyone provide insight? Thanks!
Gel #1: I tested whether boiling my samples at 98C for 5 minutes would get rid of the dots in case they were DNA clumps. It seemed to help but I still don't get a sharp band.
Precast 1%TBE
3 uL sample buffer, 14 uL sample, loaded 13.5 uL in the gel
Ran 90V for 140 min
Lanes 3 and 6 are not boiled, lanes 4 and 7 are the boiled versions of samples in lanes 3 and 6, respectively
Gel #2: I wanted to see if the dot/flame pattern was caused by too much sample, so I loaded half as much in lanes 3 and 5 as I did in lanes 2 and 4 (2 and 3 same sample, 4 and 5 same sample)
Precast 1%TBE
Ran at 90 V for 140 minutes
All samples boiled at 98C for 2 minutes
Gel #3: The plasmid preps were given to me by a colleague so I cannot speak on how they were prepared, but I thought I'd test the quality of the prep. This gel shows that. Lanes 5 and 6 are the same plasmid prep and was cut to make lane 2. Lanes 7 and 8 are the same plasmid prep and were cut to make lane 3
Precast 1%TBE gel
Gel was run at 90V for 140 minutes
1 ug of plasmid preps were loaded into wells 5-8
Has anyone seen these patterns before or know what to test next? I will be doing my own plasmid prep soon to see if I can get a better looking one as I'm thinking this may also be a cause. Please help if you can!
your gel is overloaded, and you may have salt or something left in your plasmid preps. If you're talking about the 'dots' that are particularly visible in gel 1 lane 6 and gel 2 lane 2, you don't have nice sharp bands, you have blobs. The shape of those blobby bands is typical of overloading. Rather than a sharp line as a band, you have broad bands, with a curved leading edge and a tailed trailing edge. When you run 100 ng or so, it should look a little better.
We don't call those 'dots', it's a 'tail'.
You don't give the 260/280 of the plasmid. That will give you info on the quality, too. You have crappy quality DNA. If you tell us the method of plasmid isolation we can tell you the likely contamination candidates. Your crappy DNA looks like some of the stuff I isolated decades ago, before there were kits. Is this a 'homemade' plasmid isolation protocol?
Boiling won't be the answer. That just denatures your DNA, which is not what you want to do. Don't boil DNA samples unless you want ssDNA (like in Sanger sequencing or something like that).
You can do a clean-up on the original prep, or, seriously, just re-prep the plasmid. That should take you a max of 3 days (if you have to get organized and retransform) but will be quicker than working with this crap, which may have salt or protein, or something in it. A basic rule of DNA work is "when something looks crappy, do it over rather than waste time trying to fix something that shouldn't have happened in the first place".
Good quality plasmid, as you probably know, won't leave crap in the wells (you have a little), won't give you a smear (you have a smear, although you're also overloading), and will give nice sharp bands without tails.
Good luck!
Are these "dots" running in the lanes with your samples or scattered through the gel? The issue could be your agarose gel. Make sure the agarose is completely dissolved before you pour it into your casting tray. Also, make sure it's completely solid before you remove the combs. If it's not all the way dissolved, then you'll see scattered dots in the gel. Removing combs too soon will make the edges of the wells collapse and give "smeared" bands.
Also, I've never heard of anyone boiling their plasmid after a digest.
your gel is overloaded, and you may have salt or something left in your plasmid preps. If you're talking about the 'dots' that are particularly visible in gel 1 lane 6 and gel 2 lane 2, you don't have nice sharp bands, you have blobs. The shape of those blobby bands is typical of overloading. Rather than a sharp line as a band, you have broad bands, with a curved leading edge and a tailed trailing edge. When you run 100 ng or so, it should look a little better.
We don't call those 'dots', it's a 'tail'.
You don't give the 260/280 of the plasmid. That will give you info on the quality, too. You have crappy quality DNA. If you tell us the method of plasmid isolation we can tell you the likely contamination candidates. Your crappy DNA looks like some of the stuff I isolated decades ago, before there were kits. Is this a 'homemade' plasmid isolation protocol?
Boiling won't be the answer. That just denatures your DNA, which is not what you want to do. Don't boil DNA samples unless you want ssDNA (like in Sanger sequencing or something like that).
You can do a clean-up on the original prep, or, seriously, just re-prep the plasmid. That should take you a max of 3 days (if you have to get organized and retransform) but will be quicker than working with this crap, which may have salt or protein, or something in it. A basic rule of DNA work is "when something looks crappy, do it over rather than waste time trying to fix something that shouldn't have happened in the first place".
Good quality plasmid, as you probably know, won't leave crap in the wells (you have a little), won't give you a smear (you have a smear, although you're also overloading), and will give nice sharp bands without tails.
Good luck!
Lori is correct, you are putting too much DNA in your gel (causing what you call "flames"). Generally, you want to measure the concentration of your extracted plasmids by nanodrop before you digest it. This way, you can plan your restriction digests and load a maximum of 200ng (I usually go for anything between 50ng and 200ng of total DNA).
It does look like the purity of your plasmids are not great either because you have smearing in your gels. Once again, assessing the concentration and quality of your prepped plasmids before digestion is quite important.
Boiling your samples is necessary for SDS-PAGE (vertical electrophoresis with acrylamide gels), but not for agarose electrophoresis. You may degrade further your DNA by doing so, therefore I recommend against it.
Katie A Burnette The dots ran with my samples, and are not throughout my gel
Lori M. Kelman and Martin Chenal Thank you for your insights! I was beginning to have doubts about the plasmid prep, and wasn't sure what kind of prep was done because I didn't do it. I am going to do my own and see what I come up with. I was also advised to run the gel slower at 60-70V and increase the agarose percentage to 1.5 or even 2% to try and get sharper bands. Do you think that doing that, in combination with loading much less sample, would result in sharper bands? Or can crappy quality plasmid never really give you nice sharp bands, even after a digest? Just trying to learn what mistakes cause what patterns in a gel, as I am pretty new to this (as you can probably tell)! Thanks for all your help!
1.5 and 2% gels make my ladders not run correctly, also I recommend TAE over TBE
Honestly, I run all of my agarose gels at 130V for 30mins and get great results, so I don't recommend using a lower voltage.
Sharper bands can be achieved by using thinner and wider wells. Crappy quality plasmid can give you great bands, but you may see by running a non-digested sample that a fraction of that plasmid is in a open circular or linear conformation, meaning it was cut on 1 or both strands. Look at the attached file for an example of a good quality plasmid digestion I have done (don't mind the crappy ladder).
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