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too much DNA on agarose gel

agarose gel high concentration wrong bands

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9 replies to this topic

#1 Wickerl3732

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Posted 30 January 2013 - 07:04 PM

Hi to everyone.

for purification of my PCR product I was loading a too high DNA concentration on my preparative gel, and, as one would expect, it ran way too fast and the band appeared very low. However, when my student was asking me why DNA runs too fast if you load too much, I had no reasonable explanation.

Can anyone help me out here?

#2 bob1

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Posted 30 January 2013 - 07:52 PM

It doesn't run faster if you have too much, if anything it is more likely to run slower (e,g. get caught in the well).

#3 Wickerl3732

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Posted 30 January 2013 - 08:07 PM

Well, had this problem twice in my still young post doc career and it always ran too fast. It might also happen that DNA is trapped in the well, if you load to much, but this was not the case in my hands, and this would also be related to bad 260/280.
Please see also (for example) http://www.methodboo.../agarogel.html.
But still, since it would make sense that too much DNA is caught in the well, I don't have any explanation why it runs to fast.

Edited by Wickerl3732, 30 January 2013 - 08:44 PM.


#4 neuron

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Posted 30 January 2013 - 08:23 PM

I agree with bob, it might have caught in the well. Your link is not working for me. It will be good if you could share the gel picture. Long back I faced this problem when I was learning molecular biology. But In my case the reason was that overloading would lead to spillage and I would get a streak and very faint bands of my interest. I don't know whether this could be related to your problem also.

#5 Wickerl3732

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Posted 30 January 2013 - 08:43 PM

Sorry neuron, I have no picture since I do not want to expose my preperative gel too long to the UV. But please allow me to explain a little bit more. In my case we wanted to purify a long PCR product, about 11 kb, and we need to get a certain amount (moles) out of it. So we pool 10 PCR reactions, reduce volume by Qiagen PCR purification kit, and load onto one well in preparative gel for further purification. Strong band appears at 1 kb, above a little bit of smear, and a very faint band at 11 kb. In the analytic gel, however, there is just the 11 kb band. And the same is true if I load less in the preperative gel.

It's not a problem for me, I already got my desired amount of 11kb DNA, I was just wondering why this happens...

#6 neuron

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Posted 30 January 2013 - 09:28 PM

This is really strange, If you get a very strong band at 1kb and very faint band at 11kb then how do you confirm that 1kb is not nonspecific? I mean for confirmation of PCR what do you do? You may be right also because I have never run this big DNA on gel. But what if you resolve your gel more? I really have no idea. I searched for gel pictures where 11kb band is visible, check this link-

http://www.sciencedi...014488608000241

In the picture 1kb band is not there even in the marker, either they faced the same problem as you and cropped the picture or you may be going in wrong direction.

#7 hobglobin

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Posted 31 January 2013 - 02:36 AM

The link is working without the last full stop: "http://www.methodbook.net/dna/agarogel.html
and I wonder why you cleaned up the PCR product with the Qiagen kit. It's recommended for 100 bp to 10 kb (you have about 11 kb). And you pooled 10 PCR reactions, but how many columns did you use? If only one or two, then you might have lost most of the 11 kb product as the column(s) is/are overloaded, or the columns might preferentially bind to the smaller 1 kb amplicon (a guess, not sure about this).
Anyway I'd try it without the cleaning step, as with agarose electrophoresis you also clean up the samples sufficiently. To prevent overloading, just use several lanes. This increases the work to cut out the samples, but prevents overloading.

Edited by hobglobin, 31 January 2013 - 02:37 AM.

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#8 Wickerl3732

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Posted 31 January 2013 - 04:18 AM

I should also mention that after cutting out the 1 kb band, when running it at low concentration on an analytical gel, I get the band at 11 kb.
I don't know why, but something has to disturbe the electrical field. It is very strange to me. But nevermind.
@hotglobin: right, the kit is for 1-10 kb, but it also works for 11kb. It don't think that I loose a lot of DNA. I was using 2 columns, and loaded them with 125 ul (5 reactions) each.

#9 leelee

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Posted 31 January 2013 - 07:04 PM

The PCR volume is irrelevant for overloading your column- it depends on the yield of DNA.

However, I think you are right in that you probably haven't overloaded. It is my understanding that a typical PCR yield would be about 1ug of DNA (I could be wrong), and the Qiagen columns have a maximum binding capacity of 10ug so even pooling your 5 reactions you should be under the binding capacity.

I'm still struggling with the concept that more PCR product DNA would somehow run faster on a gel. It just doesn't make any sense.
I've never heard of this before, and I've just gone and done a bit of looking around on the inter webs and I can't find any reference to this phenomenon anywhere (bar your one example, which is anecdotal).

I know you said you have excised the 1kb band from your gel, and re-run it on another gel and it then runs at 11kb. I'm baffled.

On the gels where you see the 1kb band, how well separated are your bands? I.e. do you run for a long enough time to see a few cm of separation?

#10 DrRupesh Kumar Rajput

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Posted 04 February 2013 - 04:32 AM

DNA run fast only when you increse current,
keep the voltage low and it will run slow,
very simple
it does not depend on quantity of DNA or sample,
it matter with size of DNA (bp) and respected voltage in aparatus
chears





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