Troubleshooting in pGEX Expression Vectors

Problem		Probable cause		Solution
A high basal level of expression is observed		Lack of catabolic repression of the lac promoter.		Add 2% glucose to the growth medium. This will decrease the basal-level expression associated with the upstream lac promoter but will not affect basal-level expression from the tac promoter. The presence of glucose should not significantly affect overall expression following induction with IPTG.
No GST-tagged protein is detected in the bacterial lysate		DNA sequence is not cloned in the proper translation frame in the vector.		Check DNA sequence. It is essential that protein-coding DNA sequence is cloned in the proper translation frame in the vector. Cloning junctions should be sequenced to verify that insert is in-frame. For convenience, use the pGEX 5' and 3' Sequencing Primers. The reading frame of the MCS for each pGEX vector is shown in Figure 1.1 (pGEX vectors).

Materials