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Tracy0110

Member Since 22 May 2012
Offline Last Active Jul 22 2012 09:24 PM

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Molecular cloning

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Nayeem991
23 Jul 2012 - 03:49

Topics I've Started

Digest for insert orientation

22 July 2012 - 06:01 PM

Hi,

I digetsed my plasmid (pGreen) and my inserts with the Xho and Sal enzymes and ligated them together before transforming e.coli. The insert appears to be present in the plasmid however I now need to check the orientation of the insert as Xho and Sal have the same 'sticky ends' and my cloning is therefore not directional.

Is it possible to check the orientation of my insert using a restriction enzyme digestion with these enzymes? Or should I just sequence it?

screening for GPF

22 May 2012 - 05:20 PM

I have constricted a pGreen plasmid to contain my DNA of interest and cloned the vector into e.coli. I was wondering what methods I can use to screen for GFP action in the transformed colonies on the plate other than with PCR screening?