mtDNA isolation

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rawa zahid
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mtDNA isolation

Post by rawa zahid » Tue Aug 23, 2005 7:45 am

how a mtDNA exract appears on agarose gel , as one band parallal to the expected size of a ladder marker. dose a mitochondrial pellet differ in colour according to the organism ???? i ve been searching for answers for six monthes now .can anybody help :?

Daniel Tillett
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Post by Daniel Tillett » Tue Aug 23, 2005 11:13 pm

I don't want to be harsh, but you do realise that mitochondria do vary in size depending on the organism.

To answer your question about the colour of the mitochondrial DNA, no it should not vary in colour depending on the organism (all pure DNA is colourless). However, if your DNA is contaminated with proteins and other junk then you can get a coloured pellet.

Daniel

PCR sequencing reagents

rawa zahid
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Post by rawa zahid » Wed Aug 24, 2005 9:11 am

[i think there is a missunderstanding here ,u see ,never mind the mtDNA genome size differance i still didnt get my answer. :) ,lets say that i extracted human mtDNA ,the expected size would be like 16.56bp.now do i expect this to appear as a band parallel to a similar band size in a ladder marker or it would appear near the well? that was my qustion.
also i know how DNA looks,But again that was not my qustion,i asked about the mitochondrial PELLET color not miotochondrial DNA color

Daniel Tillett
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Post by Daniel Tillett » Wed Aug 24, 2005 10:55 am

Mitochondria DNA is circular. How it runs on a agarose gel will depend on a number of factors:

1. Agarose %
2. Presence or absence of EtBr
3. Level of supercoiling of the DNA

So the simple answer is no. If you want to size your mitochondrial DNA digest with a couple of different RE and add up the bands.

I think I did answer your question about the pellet- if it is dirty it may be coloured and if so the colour will depend on what coloured contaminates are present in the organism.

Daniel

Better DNA sequencing results

rawa zahid
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Post by rawa zahid » Thu Aug 25, 2005 5:39 pm

Well, thanks for the info.this is pretty beneficial,
i would like to go for theoretical questions like if I'm to do a search on certain mtDNA regions that are more susceptible to mutations, would i go for the conserved regions or other ones?

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