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EcoRI cloning giving only inverted inserts!!??


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#1 leochicaybam

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Posted 30 September 2010 - 02:12 PM

I have a vector of 8,5kb and I am trying to clone my inserts using the EcoRI site (unique) in the MCS. The transformation worked very well and I had many colonies. However, in nearly all the colonies that had inserts, the inserts were inverted! I have to clone 5 different inserts, but after 137 mini preps I have cloned only 2!

To verify the orientation of the insert Im using an enzyme that cuts one time in the vector and one time in the insert. The difference is very clear (400pb vs 1200pb).

Someone has a clue? Or am I very unlucky?

Cheers,
leochicaybam

#2 phage434

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Posted 30 September 2010 - 02:21 PM

Very likely the insert is somewhat or very toxic to the E. coli cell in one of the orientations. Your cloning vector may have a promoter active aimed into the cloning site. You could try a different vector with terminators surrounding the cloning site, or add your own to the sequence. Lucigen makes some insulated vectors, and all of the Biobrick vectors have this isolation.

#3 leochicaybam

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Posted 30 September 2010 - 02:30 PM

Very likely the insert is somewhat or very toxic to the E. coli cell in one of the orientations. Your cloning vector may have a promoter active aimed into the cloning site. You could try a different vector with terminators surrounding the cloning site, or add your own to the sequence. Lucigen makes some insulated vectors, and all of the Biobrick vectors have this isolation.


The vector is a lentiviral vector with a SFFV promoter, generally used for hematopoietic cells. Is this promoter active in E. coli??? Maybe a "residual" activity could cause this problem?

Thanks for the answer!

leochicaybam

#4 phage434

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Posted 30 September 2010 - 03:16 PM

Cryptic prokaryotic promoters are a real possibility. But, just as likely, since you are growing it in E. coli, there are some prokaryotic promoters for e.g. antibiotic resistance active on the promoter. These may read-through into the cloning site.




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