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how to seperate double-digestion products on agarose gel


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#1 Biogareth

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Posted 27 September 2011 - 11:13 AM

After double digestion of my target plasmid, there are two products with similar sizes: one is 2.2 kb, the other is 2.3 kb. I have used 120 Voltage, 45 min, but these two bands were not be seperated with 1% agarose gel.
So I wonder is it possible they can be seperated?
Any suggestions are welcome!

#2 hobglobin

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Posted 27 September 2011 - 12:04 PM

You can try a different agarose type such as Nusieve with higher resolution and/or using a different buffer system (e.g. Lithium Borate instead of TAE). A longer gel also helps of course. Or use different enzymes so that you get fragments with a larger difference, if it's possible.
One must presume that long and short arguments contribute to the same end. - Epicurus
...except casandra's that belong to the funniest, most interesting and imaginative (or over-imaginative?) ones, I suppose.

#3 Biogareth

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Posted 27 September 2011 - 12:31 PM

You can try a different agarose type such as Nusieve with higher resolution and/or using a different buffer system (e.g. Lithium Borate instead of TAE). A longer gel also helps of course. Or use different enzymes so that you get fragments with a larger difference, if it's possible.


Thank you for your fast reply!
BTW, do you think it can be solved by increasing agarose concentation, like 2%? Becuase it will be more convinient.
Thanks!

#4 bob1

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Posted 27 September 2011 - 02:49 PM

You would be better off running it for longer rather than changing the % gel - 2% just means that they will migrate slower for bands of that size.

#5 phage434

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Posted 27 September 2011 - 03:47 PM

There may be an enzyme that will cut the the fragment you don't want.

#6 Biogareth

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Posted 28 September 2011 - 05:21 AM

There may be an enzyme that will cut the the fragment you don't want.


Thanks, it looks it is a good idea.
But I am wondering restriction enzyme can really 100% cut the fragment which I don't want, otherwise, this contaminatant will influence my futher ligation with another vector.

#7 Biogareth

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Posted 28 September 2011 - 05:41 AM

You would be better off running it for longer rather than changing the % gel - 2% just means that they will migrate slower for bands of that size.


I just tired to run with 1 hour at 130 voltage with 1% agarose, but these two bands still can not be seperated.
Attachment is the picture.

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#8 leelee

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Posted 28 September 2011 - 07:11 AM

How about running at a lower voltage but overnight? I can usually get pretty good separation of larger bands for my RFLPs. I use 0.8% agarose in 0.5 TBE buffer and run overnight at 70V (or longer even), using a 23cm gel tray.

#9 leelee

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Posted 28 September 2011 - 07:11 AM

Another idea would be to design primers to amplify the region you want?

#10 Biogareth

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Posted 28 September 2011 - 07:26 AM

How about running at a lower voltage but overnight? I can usually get pretty good separation of larger bands for my RFLPs. I use 0.8% agarose in 0.5 TBE buffer and run overnight at 70V (or longer even), using a 23cm gel tray.


It is a good idea!
I wonder how big your bands can be seperated in this case? My two fragments: one is 2.2kb, and the other is 2.3kb.
Thanks!

#11 allynspear

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Posted 28 September 2011 - 07:34 AM

phage434 is 100% correct on this issue. No matter how hard you try, fragments that close in size will almost never be resolved, especially when running the quantity of DNA you need to get good yield from gel purification. There will also be a tendency for DNA from one band to streak into the other, even if you can't see it by EtBr/UV. Your concern about the efficiency of cutting is not significant, as even 90% efficiency of cutting, combined with your electrophoresis separation will give you at least a 20:1 ratio of correct fragment to incorrect. You should still be able to clone from this, and I would wager that your enzyme will cut near 100%. I've done this tons of times and it works every time.

Best of Luck.

#12 hobglobin

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Posted 28 September 2011 - 09:00 AM


How about running at a lower voltage but overnight? I can usually get pretty good separation of larger bands for my RFLPs. I use 0.8% agarose in 0.5 TBE buffer and run overnight at 70V (or longer even), using a 23cm gel tray.


It is a good idea!
I wonder how big your bands can be seperated in this case? My two fragments: one is 2.2kb, and the other is 2.3kb.
Thanks!

usually it should not work well as the bands have also a lot of time to diffuse and you will have much broader bands in the end....the shorter the time running the better. To overcome the problem that then the gel length cannot be used completely (long trays) you can use a buffer like Lithium borate buffer that can be used with high voltages without getting too warm.
One must presume that long and short arguments contribute to the same end. - Epicurus
...except casandra's that belong to the funniest, most interesting and imaginative (or over-imaginative?) ones, I suppose.

#13 leelee

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Posted 28 September 2011 - 06:21 PM

usually it should not work well as the bands have also a lot of time to diffuse and you will have much broader bands in the end....




Interesting, I've never seen this, doesn't happen for me. Perhaps if the voltage was excessively low you might see that? But then again I've even run my gels over the weekend at as low as 20V (after an initial run of a couple of hours at 70V) and my bands are still able to be clearly resolved. I wonder, how can the DNA diffuse if you keep the voltage on?

Having said that, for your purpose, Biogareth, I don't know if this will work for your case (sorry didn't properly read you the first time so didn't realise the size difference was so small), as although you might be able to tell difference between bands of similar size, they are probably still too close to be certain you haven't got any contamination from the other band when you excise from the gel (particularly when you consider the points allynspear makes).

I tend to agree with phage and allynspear and think that the use of another RE is the way to go.

What are you using your fragment of interest for?

#14 hobglobin

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Posted 29 September 2011 - 10:40 AM

usually it should not work well as the bands have also a lot of time to diffuse and you will have much broader bands in the end....




Interesting, I've never seen this, doesn't happen for me. Perhaps if the voltage was excessively low you might see that? But then again I've even run my gels over the weekend at as low as 20V (after an initial run of a couple of hours at 70V) and my bands are still able to be clearly resolved. I wonder, how can the DNA diffuse if you keep the voltage on?

Some time ago we wanted separate PCR fragments with standard agarose gels (large chambers) as far as possible, and one idea was to run it over night and use the gel distance as far as possible...anyway we were surprised that the bands were much broader in the end and the resolution decreased a lot...So we didn't do it again after some attempts and attributed this to diffusion. In some articles and protocols about agarose gel electrophoresis you can find this observation too and the recommendation to run the gels not too long.
One must presume that long and short arguments contribute to the same end. - Epicurus
...except casandra's that belong to the funniest, most interesting and imaginative (or over-imaginative?) ones, I suppose.

#15 leelee

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Posted 29 September 2011 - 06:26 PM

Fair enough then.
I guess if its not a problem for us, I won't worry about it Posted Image




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