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extra unknown band after restriction digestion?Moderator: BioTeam
12 posts • Page 1 of 1
extra unknown band after restriction digestion?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.
Re: extra unknown band after restriction digestion?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.
Re: extra unknown band after restriction digestion?I forgot to say: yes, there was not any smear. Only a band bigger than 10 kb.
Re: extra unknown band after restriction digestion?Yes, I checked it. It also have the same extra band
Re: extra unknown band after restriction digestion?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.
Re: extra unknown band after restriction digestion?<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>
Re: extra unknown band after restriction digestion?attached other format of the view
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.
Patrick
Science has proof without any certainty. Creationists have certainty without any proof. (Ashley Montague)
12 posts • Page 1 of 1
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