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Is it possible to separate DNA with only 10bp difference?


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#1 hello world

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Posted 29 December 2010 - 09:40 PM

I am trying to separate DNA with 803bp and 813bp.

Can anyone tell me whether this is possible and the wehat % of agarose/polyacryamide should i try?

This step is utmost important because if i fail in doing so, i may have to do single digestion for plasmid and gene and pick colonies forever (dont mention 5-phosphatase and terminal transferase, we dont have it in lab).So, plz help~

by the way, i need to isolate the 803bp DNA framgent from the gel in the later steps. So, i need a lot amount of fragments.

Edited by sexymonkey, 29 December 2010 - 10:23 PM.


#2 pDNA

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Posted 29 December 2010 - 11:52 PM

sounds not that easy but could be possible with a lot of patience :)
never did it on my own but there is this reference, that says it is possible with low conductive medium:
http://www.biotechni...04_O_37093a.pdf

Hope this helps!
Good luck!

Regards,
p

#3 hobglobin

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Posted 30 December 2010 - 03:05 AM

I'd also try out with a lithium borate buffer (described in the paper above) and use longer gels for a longer migration distance. If you use agarose I'd try a 1.2 - 2% gel.

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#4 atwood

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Posted 30 December 2010 - 08:07 AM

hihi~ i have the similar question too. Thanks for answering. However, i dont pretty understand the principle.

From what i know, high salt concentration will usually generate very high heat, for example IEF. i dont see any special equipment required in the paper, so, how it possible that no temperature was found in the experiment? Do i miss something in the paper?

Moreover, in the paper, it run in a high voltage such as 400V~200V.
From physics lesson, we have learnt V=IR, For high V and low I, this represent that there is high resistance. High resistance should generate high heat.

Lastly, this method seems to be so efficient that it can replace glass capaillary electrophoresis.
Hence, is the method stated in this paper really work?




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