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Unusual band in transformants


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#1 genesquirt

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Posted 07 January 2011 - 12:16 AM

Greetings, I would like to ask your opinion about my little problem.

I've been working on ligating, identifying and sequencing an unknown plasmid from a bacteria.
I've done all the necessary step and come to the point where I am sequencing my plasmid vector.
I have ligated an unknown plasmid DNA with pUC19 and transformed it in DH5α. After selecting the colonies of the transformants, I put them to LB broth with ampicillin.

When I check the plasmid bands, one of them has 3 bands (looks weird for a transformant). The three bands were located in this manner approximately 10kb, 8kb and 3kb in size. This was before digestion. By the way I used EcoRI restriction endonuclease for this experiment. So after digestion with EcoRI, 2 bands which is located at approximately 10kb and 8kb were lost after the digestion.

I would like to ask your opinion what had happen? Is it still okay to be sequenced?

Thanks in advance!

If you need to clarify anything. Please don't hesitate to post.


#2 ElHo

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Posted 07 January 2011 - 01:17 AM

Usually there are three major bands when separating uncut plamid DNA by gel electrophoresis: Relaxed, linear and supercoiled, the last one migrating fastest. Thatīs maybe the reason why this band will not dissapear after digestion.
Do you get any additional smaller bands after digestion?

#3 HomeBrew

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Posted 07 January 2011 - 04:35 AM

ElHo's right -- what you're seeing in your undigested sample is three different forms of the same plasmid. As these different three-dimensional forms occupy varying amounts of space, they migrate through the gel at different rates, producing what appears to be three different bands (this is why you can't estimate their sizes by comparison to a linear standard). Digestion with a single-cutting restriction enzyme resolves all these different forms into linear molecules; this sample thus appears as a single linear band whose size can be estimated by comparison to your linear standard.

This is a well-known phenomenon -- you can proceed with sequencing.




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