A PCR result confirmed the presence of an insert in the recombinant plasmid (pGLO vector), but there was no BSA protein expressed after arabinose induction.
1. What would be the source of my problem?
2. What do I ensure in the methods to make sure it doesn't happen again?
I have no experience regarding protein expression using pGLO, but here are some of troubleshooting that I have about protein expression using IPTG.
- There might be a problem with the inducer, try using a freshly made inducer.
- Check the sequence, make sure there is no stop codon in front of the gene
- Without purifying, run the lysate in SDS-PAGE just to make sure there is difference between induced and non-induced one
You're going to have to give a lot more information to get a helpful answer. What did you use for controls? There are a LOT of steps between PCR from a ligated vector, successful transformation, and protein expression. What is the last step that you have evidence to show it worked properly?
I'd suggest you talk with your mentor. This is way too vague to get a good answer from folks who haven't seen you in the lab.
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