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Restriction Digest Assignment Difficulty

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Restriction Digest Assignment Difficulty

Postby Cist » Sun Oct 31, 2010 12:25 am

I am having trouble with one of the questions in my restriction digest assignment, a classmate and I have been thinking about it for days but we can't figure it out. Can anyone help?

A large number of 10kbp plasmids are digested:

Endonuclease / Fragments
BamHI / 10
EcoRI / 10
PstI / 2.7 , 7.3
NotI / 10
SstI / 4.8 , 5.2
TaqI / 6 , 10 , 17 , 23

It then goes on to do double digests with varying combinations and I drew the circular restriction map. The 2nd part of the question asks to identify anomalies and say why they could happen.

Obviously the anomaly is that somehow a 10kbp fragment is digested into 6, 10, 17, and 23 fragments. Also, for example, if you mix TaqI with PstI you get 2.7 and 7.3 fragments. This leads me to believe that the TaqI does not cut at all. But I have no idea whats going on with the 6, 10, 17, and 23.

Thanks!
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Postby JackBean » Sun Oct 31, 2010 9:03 am

http://www.biolib.cz/en/main/

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Postby Cist » Sun Oct 31, 2010 1:42 pm

Thanks for you help. I've searched around that site but I am not sure what I am looking for. I am thinking it might have something to do with the high optimal temperature...
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Postby JackBean » Sun Oct 31, 2010 4:50 pm

OK, I think I got the answer :)
http://en.wikipedia.org/wiki/Plasmid#Conformations

the size is not actuall size of some linear fragments, but the plasmid was NOT cut by TaqI and is still intact :)

(do you have some double digest with TaqI? Does it differ from digest by only the other RE?)
http://www.biolib.cz/en/main/

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Re: Restriction Digest Assignment Difficulty

Postby Cist » Sun Oct 31, 2010 6:40 pm

So...

The gel electrophoresis is more or less designed for linear DNA molecules? So the specific size, shape, and charge density of the plasmid moves faster through the gel giving a false impression that it is bigger than it actually is? And the cross-linking of plasmids gives the impression that there are multiple fragment? (i.e. 1 plasmid looks like a 23 fragment, 2 cross-linked plasmids look like 17 fragment, 3 cross-linked looks like 10, etc.) and therefore there is nothing specific about the numbers 6, 10, 17, 23, they just happen to be what the false results look like.

I'm really racking my brain here, that's the best I've got.

and thanks for your time!
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Re: Restriction Digest Assignment Difficulty

Postby JackBean » Tue Nov 02, 2010 5:57 pm

Cist wrote:So...

The gel electrophoresis is more or less designed for linear DNA molecules?


Not exactly, the point is, that the molecules move in accordance to their volume and shape. So, if you take two 4 kbp circular plasmids, one will supercoil and will be thus smaller, while the relaxed one will be kind of large and thus will travel slower.

Did you have some other digest including TaqI? (like double digest of EcoRI and TaqI or similar?)
http://www.biolib.cz/en/main/

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