I have been trying to express my construct for the last couple of weeks but after several attempts, it's still failing although similar parts (five inserts for golden gate assembly) have been used by my colleague in past and it worked. I want to know if I am doing something wrong with my ligation reactions or digestion of parts is working fine despite I have checked protocols several times. Here, I would like to request the expertise if anyone can suggest options which I can use to make sure I following the right path.
Thank you in advance.
So are you having issues with the cloning or is the final construct not functioning?
First you need to be sure that your clone is correct, preferably by sequencing the complete insert.
Secondly, if you are certain the plasmid is correct and you are not getting expression, try to transform that plasmid again into fresh cells and screen a number of colonies to see if any of them express your protein.
I agree with Michael.
Some constructs are simply not stable - either because they contain internal homologies or because they are harmful to the cells.
Sequencing is the best way to check, but doing a plasmid extraction and checking the insert size by restriction digestions will often reveal any anomalies.
Hi Robert Adolf Brinzer , I am having an issue with the final construct even after multiple attempts and I have also tried different concentrations with stocks. However, a similar construct has been worked on earlier, and successfully expressed by one of my colleagues. So, I am interested in exploring what could be the reason behind the failure of this construct.
Have you done PCR on the cDNA to troubleshoot if it is at the transcription or translation side of expression? Some proteins are highly regulated so can be sent down lysosomal degradation paths if overexpressed.
Thank you Michael J. Benedik I have tried screening different colonies as well but no outcome has been observed. As per your suggestion, I might try to sequence the construct to double-check and hopefully, I will get some kind of explanation.
Hi Andrew Jenkins I will try to follow your advice. Although the similar parts have worked earlier it's just not working anymore and similar with other parts. Do you think it has something to do with restriction enzyme activity? As transformation is working okay because I see a healthy growth of BL21 with Kanamycin resistance in all of my cultures.
Difficult to say whether the restriction enzyme is the problem, but a quick agarose gel should determine whether it is linearising your vector and/or cutting your insert correctly.
Good luck. Cloning can be very difficult to troubleshoot. There are so many steps and all of them can go wrong!
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