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
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