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Specialty Chemistry Forums => Biochemistry and Chemical Biology Forum => Topic started by: 2810713 on March 09, 2005, 07:02:57 AM

Title: DNA electrophoresis
Post by: 2810713 on March 09, 2005, 07:02:57 AM
Hi all,  :D
  I have seen marks denoting the number of nucleotides along the gel which actually denote the molecular weight. But , do the polynucleotides with same number of nucleotides have same molecular wt.always? I think , they would not , as the proportions of A ,T ,G & C would not be the same always.Thus , they will get separated during electrophoresis . So, how can we have a common mark denoting the nucleotide number for such polynucleotides as they would be apart from each other . Can we have only sizewise separation of molecules in electrophoresis? If yes , then ,I think , such polynucleotides may have same mark as the size-difference between them seems to be lesser.

Please help..

Title: Re:DNA electrophoresis
Post by: Mitch on March 11, 2005, 01:03:29 AM
I've only seen it done by weight not by number of nucleotides.
Title: Re:DNA electrophoresis
Post by: 2810713 on March 11, 2005, 04:49:57 AM
  I am a junior college student and have seen it in our Bio. lab.
 only once, I've also , solved problems with such a diagrams.

Title: Re:DNA electrophoresis
Post by: Donaldson Tan on March 11, 2005, 08:52:52 AM
junior college student? are u studying in singapore?
Title: Re:DNA electrophoresis
Post by: 2810713 on March 12, 2005, 09:58:33 AM

No, Bharat[ INDIA]

I think, U R in singapore???

Title: Re:DNA electrophoresis
Post by: savoy7 on March 15, 2005, 11:12:33 PM
Old school -

I haven't done Recombinant work in years (+ decade), so I don't know how they do it now.

In the past:

small pieces of DNA can be separated using polyacrylamide gels( 10 - 500 bases) - single strand
due to the small size of the pores, these can't separate large pieces of DNA

agarose gels then come into play - up to certain point (300 to 10 maybe 30000 bases),  extremely large pieces of DNA ( if memory serves me right 30000 to 2 million bases) needs a process called pulsed-field gel electrophoresis

Since DNA is negative and all of these gels have pores, etc - the DNA is separated by size (bases).  To determine the size, a standard is used.  One buys them - some DNA cut by a restriction enzyme like BamHI, EcoRI or TaqI.

We would know the sizes of the DNA pieces in the known.  We create a log plot off that data based on size (# of bases).  

then we could estimate the sizes of our sample DNA.

Problem - potentially 2 pieces of DNA could be the same size.  In protein land, we used to use a 2 dimensional gel, I wonder if they do that now to eliminate this problem.  

As for using weight, size does relate to weight - but purines and pyrimidines do have different weights - on a large piece of DNA that weight may not make much of a difference, but on a very small piece of DNA ( a couple of nucleotides) it does.

I apologize if this post is information you already know, I don't know your background on this material.

Title: Re:DNA electrophoresis
Post by: 2810713 on March 16, 2005, 07:05:45 AM
 I also, expected that but how to ba certain about it. The last part was useful- the difference is neglegible, also, in most cases one deals with DNA from one species , so there the
A+T/ G+C ration is constant so there is naturally there is further less difference bet. molec. wt.s .

thanks, I didn't know about pulsed feild gel electro phor.
 I will search now. thanks.