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Cloning synthesised genes

Molecular Biology

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#1 Almenis

Almenis

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Posted 21 July 2013 - 03:29 AM

Hi everybody!

I ordered some genes recently to a synthesis company that I received in pUC vectors. I am having quite a lot of troubles cloning them into retroviral vectors, I have changed multiple parameters figuring out troubleshooting at different steps but nothing is really working beyond the ligation step. The fact is that the ligase works and we are able to do some other cloning with material not coming from synthesis. All the clonings are very simple designed (e.g. HindIII-XhoI digestion, nice digestion and purification, etc).

I am always getting kind of pUC empty vectors in the final minipreps I am beggining to wonder if the starting material is clean enough to get a good efficiency cloning (I did not sequence the plasmid neither clones at the very beggining and that might be a mistake?)

I thank any advise about that!!

Maria

#2 bob1

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Posted 21 July 2013 - 03:43 PM

If this is pUC57, then there isn't an Xho1 site on there at all, unless you got one inserted on your gene during synthesis.

You could easily transform the synthesized gene (in plasmid) and do some preps so that you can be assured of the DNA quality.





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