extra unknown band after restriction digestion?

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genetherapy
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extra unknown band after restriction digestion?

Post by genetherapy » Mon Jul 16, 2012 3:24 pm

I have a vector size 4775 bp and insert 500 bp. I ligated them before and yesterday I digested with related restriction enzymes for my experiment. But interestingly I saw 3 bands. One is insert(500 bp) the other is vector (4775 bp) and there is an other band bigger than 10000bp (My marker's bigger size is 10000). Today I digested again and saw the same bands. I use stock pellets. I mean, I purify plasmid DNA from bacterial pellet and then I do restriction digest. Could it be the bacterial DNA if I could not purify well?? But E.coli DNA is 4 million bp. Could anyone give some advice.
Greatly appreciated.

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JackBean
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Post by JackBean » Tue Jul 17, 2012 10:29 am

If you'd digested E.coli gDNA, you would probably get more than one band. Are you sure there was no background smear?
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.

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Re: extra unknown band after restriction digestion?

Post by genetherapy » Tue Jul 17, 2012 6:48 pm

I didn't digested e.coli DNA. I purified plasmid dna from e.coli and then digested plasmid DNA. Could it be the e.coli DNA if I could not purify plasmid DNA from bacteria (top 10 e.coli) well. my biggest marker is 10 kb and this extra band is bigger than 10 kb.

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Re: extra unknown band after restriction digestion?

Post by genetherapy » Tue Jul 17, 2012 6:49 pm

I forgot to say: yes, there was not any smear. Only a band bigger than 10 kb.

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JackBean
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Post by JackBean » Wed Jul 18, 2012 8:01 am

Did you check uncut plasmid?
http://www.biolib.cz/en/main/

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Re: extra unknown band after restriction digestion?

Post by genetherapy » Wed Jul 18, 2012 11:48 am

Yes, I checked it. It also have the same extra band

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Re: extra unknown band after restriction digestion?

Post by genetherapy » Wed Jul 18, 2012 12:18 pm

And yesterday I loaded little amount and looked the gel,did not see any smear but today a loaded whole restriction reaction to gel and checked it and realised have a upward smear. Also have the extra band. Downward smear means degredation but what does upwaard smear. Still thinking about bacterial contamination??? I really need help. Attached the gel view.
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JackBean
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Post by JackBean » Wed Jul 18, 2012 12:37 pm

But otherwise it's distinct from the cut DNA, right?

The picture is not working. Try different format or upload it directly e.g. to tinypic.com
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Re: extra unknown band after restriction digestion?

Post by genetherapy » Wed Jul 18, 2012 1:49 pm

<a href="http://tinypic.com?ref=123oztv" target="_blank"><img src="http://i49.tinypic.com/123oztv.jpg" border="0" alt="Image and video hosting by TinyPic"></a>

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Re: extra unknown band after restriction digestion?

Post by genetherapy » Wed Jul 18, 2012 5:02 pm

attached other format of the view
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Post by genetherapy » Mon Jul 23, 2012 3:30 pm

Nobody has an idea??

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Post by canalon » Thu Jul 26, 2012 2:57 am

NOt sure what we are looking at in each lane. But my guess, since it appears that you have loaded a ton of DNA in your gel (considering the look of the band), would be trace amount of genomic DNA.
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Science has proof without any certainty. Creationists have certainty without
any proof. (Ashley Montague)

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