Hi,
I am working in recombinant protein expression in E. coli BL21(DE3). Earlier I got the over-expressed proteins at 70 kDa. And I stored my construct as glycerol stock in -80 C. Now, I am reviving my cultures and suddenly I am not getting any protein expression under IPTG induction. I was cloned and expressed in pGEX-4T2 vector. I started my experiment with fresh cloning and expression, even though I am not getting any such protein expression from my previous results. Please anyone can suggest me, why I am not getting protein expression and what kind of troubleshoot needed to over come this problem?
Anyone Please help me..
Did you perform a sequencing to check if your insert is inside ? It is a good idea to use primers that overlap half with the plasmid and the other half with the insert. It is a fairly large insert so also use primers in the middle of your insert.
Plate your old glycerol stock towards single colonies, perform colony PCR with the primers you use in sequencing (to check if the insert is inside). Plate positive colonies again and start from fresh colony for preculture and IPTG induction afterwards.
Let me know what happens !
kind regards
i hope you may lost the plasmids. you should repeat the experiment again...
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