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no protein expression


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#1 amita22

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Posted 11 November 2013 - 09:15 AM

Hi,

I am facing issues in expression of my gene of interest. I have custom synthesized a single fragment (AseI -- lacI-pLacIq-pTac-SD-GOI-stop codon-terminator ---EcoRI). the pLacIq and ptac are in reverse direction with a gap of 6 base pairs. I have cloned this fragment into a vector. after digestion with enzymes, I am getting the right fragments also. I am even able the grow the transformed cells in the presence of 100ug/mL Amp BUT there is no protein expression even after inducing with 1mM IPTG (have tried 50/100/200/500/1000um IPTG). I am unable to guess why is this happening, is this happening because pLacIq and pTac are too close???. Please suggest.



#2 jerryshelly1

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Posted 11 November 2013 - 09:51 AM

Have you double checked to make sure you cloned everything in the correct frame?



#3 amita22

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Posted 11 November 2013 - 11:27 AM

Have you double checked to make sure you cloned everything in the correct frame?

well.. I havecpasted the vactor map for ur review.

 

pATM1Map.png



#4 jerryshelly1

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Posted 11 November 2013 - 12:10 PM

That doesn't really help. How did you design your primers for insertion into your vector. Can you paste or attach the sequence that you used to engineer your primers for RE digestion and subsequent ligation?






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