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Contamination


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5 replies to this topic

#1 euchromatin

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Posted 06 July 2010 - 06:53 PM

Dear labrats,

I have recieved a plasmid from Addgene. After isolation the plasmid from E.coli cells by Qiagen maxi-prep kit, an unpredicted band was appreared around 500bp size. At the first, I thought the band was RNA contamination. However, the band was not disappreared after retreating RNase A and did not look like a RNA band becase the band was sharp.

Do you guys think this is RNA contamination or DNA contamination? If this is DNA contamination, where it has come from and how to remove this?

I think someone has similar experience about this. Please let me know how to solve this problem? :P Thank you guys.

#2 leelee

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Posted 06 July 2010 - 09:50 PM

What do you mean unexpected?
Have you digested your plasmid with an RE? Otherwise, you can't compare circular plasmid with a linear DNA marker. The band you are seeing could be one perhaps super-coiled plasmid which can run quite easily through a gel....
Could you put up your picture?

#3 euchromatin

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Posted 07 July 2010 - 01:04 AM

I have already cut the circular plasmid at a single site by a restriction enzyme, appearing a single linear band. But, the unexpected band still remained at the same position.

I attached the picture. Lane #8 and #9 are uncut circular plasmid DNAs. Do you think the lowest band is a supercoiled plasmid DNA?

I have never seen that kind of supercoiled bands. Please give me more comments about this. Thank you.

Attached Thumbnails

  • 1.JPG

Edited by euchromatin, 07 July 2010 - 01:12 AM.


#4 saraarasus

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Posted 07 July 2010 - 05:15 PM

If you don't want it. cut the the part that you want and purify it from the gel.
I mean if you have never eaver see such a band with the plasmid, just get ride of it. It can be any thing. degrated part or contamination??????????????????????

Dear labrats,

I have recieved a plasmid from Addgene. After isolation the plasmid from E.coli cells by Qiagen maxi-prep kit, an unpredicted band was appreared around 500bp size. At the first, I thought the band was RNA contamination. However, the band was not disappreared after retreating RNase A and did not look like a RNA band becase the band was sharp.

Do you guys think this is RNA contamination or DNA contamination? If this is DNA contamination, where it has come from and how to remove this?

I think someone has similar experience about this. Please let me know how to solve this problem? :lol: Thank you guys.



#5 perneseblue

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Posted 07 July 2010 - 05:48 PM

I am a bit unclear over a few points

is that band present in undigest plasmid DNA?

is this band present only when you digest your plasmid with restriction enzyme (the enzyme that you used).

Does this band disappear if you use a different RE?

My main concern is that this band, is actually part of the plasmid that was cut out. Meaning the sequence map you have, or the plasmid you were given, is the wrong thing. Plasmid mix-ups, and improper construction do happen.
May your PCR products be long, your protocols short and your boss on holiday

#6 euchromatin

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Posted 07 July 2010 - 07:44 PM

Thank you for your comments. I need to make sure that the band exists in undigested plasmid. Even if I digest the plasmid by another REs, the band does not disappear.

I'm trying to prepare plasmid DNA from another single colonies. If the band still exists in re-prepared plasmid DNA, I will purify upper plasmid DNAs that I want from agarose gel as saraarasus' recommendation.

Thanks for you guys' help.




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