NovaBlue(DE3) versus BL21(DE3): transformation problems? - (May/31/2008 )
This is my first post...I'm now desparate. I have been attempting to transform pET15b+insert (from bacterial protein) into BL21(DE3). There are no colonies. The insert has been sequenced, is in frame, and PCRs up. I have no problems getting colonies if this vector is transformed into XL1 cells, NovaBlue cells and NovaBlue(DE3) cells. I believe the BL21(DE3) cells are viable as they are new and the positive control (test plasmid) produced colonies. A postdoc in my lab also used the BL21(DE3) cells with success. The protein I want to express is not toxic and the vector is only ~6500 bp. To confirm that I am not making any errors, I had the aforementioned postdoc try the cloning from the start (PCR to digestion to ligation to transformation). He has also experienced the same result. Any reason as to why we can get NovaBlue(DE3) colonies and no BL21(DE3) colonies? I suppose as long as I can express my protein in NovaBlue(DE3), then this is a non-issue; however, we are still bewildered.
Are you transforming the BL21 cells from a ligation reaction, or is it purified plasmid?
If you are transforming using a ligation product, I would suggest putting it into XL-1 Blue first (which you say you had success with) and then perform a purified plasmid prep (I recommend the Qiagen mini prep kit)
The pET system is a low copy plasmid, and you will need to purify about 10mL's of overnight LB+ antibiotic culture to get enough plasmid.
After you get the purified plasmid from XL-1 Blue, confirm it by either restriction digestion or PCR. Use the purified plasmid prep to transform your BL21 E. coli.
Thanks for your reply. Yes, I have transformed the ligation product first into XL1, then performed a plasmid miniprep (Qiagen) of a 5 mL overnight growth. Then, I checked the insert was present by PCR using the purified plasmid as the template. The insert PCRs up. Then the purified plasmid was transformed into both NovaBlue(DE3) and BL21(DE3). Only NovaBlue(DE3) produced colonies. Yep...a bummer.