nondenaturing PAGE to separate dna - (Jun/29/2009 )
hi all
Im facing problem with nondenaturing gel electrophoresis , i have to resolve 2 restriction digestion fragments that are of size 2021bp and 2109 bp ,the difference between them being 88bp. i have been trying to resolve them in 5% gel but each time i run the gel i get different result.someimes the fragments separate and sometimes they dont, again at times smiling effect is obseved. im confused and have no solution to this .
i run the gel at 6V/cm.
Is there any way to solve this problem.plz can anyone help?
deespike on Jun 29 2009, 11:27 AM said:
Im facing problem with nondenaturing gel electrophoresis , i have to resolve 2 restriction digestion fragments that are of size 2021bp and 2109 bp ,the difference between them being 88bp. i have been trying to resolve them in 5% gel but each time i run the gel i get different result.someimes the fragments separate and sometimes they dont, again at times smiling effect is obseved. im confused and have no solution to this .
i run the gel at 6V/cm.
Is there any way to solve this problem.plz can anyone help?
Just one thing to let you know that in mol biology protocol book, they said to do NAtive PAGE (3%) if size of DNA bands are >= 1000 bp. and 5% is for 100-500 bp fragments.
As your fragments are much bigger than this, you might try agarose instead of native PAGE.
thanks
epigenetics on Jul 1 2009, 12:59 AM said:
deespike on Jun 29 2009, 11:27 AM said:
Im facing problem with nondenaturing gel electrophoresis , i have to resolve 2 restriction digestion fragments that are of size 2021bp and 2109 bp ,the difference between them being 88bp. i have been trying to resolve them in 5% gel but each time i run the gel i get different result.someimes the fragments separate and sometimes they dont, again at times smiling effect is obseved. im confused and have no solution to this .
i run the gel at 6V/cm.
Is there any way to solve this problem.plz can anyone help?
Just one thing to let you know that in mol biology protocol book, they said to do NAtive PAGE (3%) if size of DNA bands are >= 1000 bp. and 5% is for 100-500 bp fragments.
As your fragments are much bigger than this, you might try agarose instead of native PAGE.
thanks
@epigenetics: But deespkie's fragments just different in ~80bp, which can't be detect by agarose.
@deespike: I did the same thing in the past with 6% nondenaturing PAGE, but I'm not sure you're using the same procedure or not. I remember that if I was too hurry to run the gel with short time for solidification and pre-running, it's easy to see the smiling effect. With this difference in size, you might have to run at least 15 cm to make them separate. Beside that, you might reduce the loading amount, because the thinner the band, the better distinguishable distance (if you afraid of the low sensitivity of staining, using silver staining is the good choice).
hi
thanks for your suggestion.Im trying out all possible ranges of acrylamide concentration but there is still problem with the separation.i run till bromophenol blue goes out of the gel.
what is the voltage in which you had achieved your separation?
deespike on Jul 2 2009, 05:52 PM said:
thanks for your suggestion.Im trying out all possible ranges of acrylamide concentration but there is still problem with the separation.i run till bromophenol blue goes out of the gel.
what is the voltage in which you had achieved your separation?
I don't remember the voltage I used. But I don't think the voltage is matter, because it just relates to the immigrating speed of your DNA. What if you actually have only ONE BAND?