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Advice needed to subclone big inserts (6 kb) into pET system

Subcloning big insert pET system

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#1 kpchai

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Posted 27 February 2013 - 09:52 PM

Good day,

I need some advices from the experts here.

I need to subclone an amylopullulanase (~6 kb in size) into an expression vector, preferably pET system with His tag at both termini.

Any suggestion on the expression vector? Vectors other than pET systems are also welcome.

However, pET systems are normally >5 kb, so pET+insert would be >10 kb and E. coli may produce inclusion bodies.

Any suggestion on the choice of host?


Thanks in advance for the advices.

#2 Curtis

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Posted 01 March 2013 - 09:51 AM

I think you need to do electroporation. 10 k is almost on the borderline for chemical competent cells.

#3 neuron

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Posted 05 March 2013 - 03:08 AM

It can be done in pET vector, I found this paper-

http://www.plosbiolo...al.pbio.0060163





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