Hi. I am tring to express recombinant protein. I obtained the nucleotide sequence of the protein I want to express through cDNA cloning and obtained the ORF sequence of the protein inserted into the plasmid. I designed primers to introduce restriction enzyme sites and confirmed the desired sequence (primer sequence with restriction enzyme sites and the protein's ORF) through PCR and sequencing. The restriction enzymes used are Nde1 and BamH1.
I treated the PCR product and pET28a (a vector for recombinant protein expression) with restriction enzymes. The reaction conditions, including buffer and temperature, were determined according to the manufacturer's protocol, with a reaction time of two hours (manufacturer's recomandation is 1 hour). BamH1 was processed first, followed by PCR purification of the vector and insert. Similarly, Nde1 was processed, followed by agarose gel purification. The purified DNAs were ligated using Takara Mighty Mix and transformed into E.coli BL21 strain. The TF strain was spread on Kanamycin LB plates. The Kanamycin concetration is 50ug/ml. Although there were not many colonies, I obtained a few colonies after about two days. Colony PCR was performed on the obtained colonies, and bands of the desired size (the same size as the PCR for introducing restriction enzyme sites) were confirmed.
Therefore, we attempted to recover the plasmid from BL21 and hoped to confirm it again through sequencing before expressing the protein. However, there is a problem. Surprisingly, plasmid extraction from BL21 does not succeed. Typically, when we extract cloning plasmids in our laboratory, we obtain around 200-600 ng/ml. However, in this case, it is below 50 ng/ml. Despite ignoring the recommended concentration of 100 ng/ml by the sequencing company, we proceeded with sequencing, but no results were obtained. We have tried the described process several times, but we consistently encounter the same issue. Plasmid extraction seems impossible. By the way, the Nde1 site of pET28a exists in the T7 tag region. I am aware that this is necessary for purification. However, since I am going to use a 6xHis tag, I intended to remove it. I am suffering greatly due to these results. Thank you very much for taking the time to read through the lengthy text. I truly appreciate it. Is there something I have overlooked in this process? I seek your professional advice and will strive to follow it as much as possible.
Because BL21 expresses the EndA nuclease, sometimes it can be difficult to get good plasmid DNA unless you take special precautions, it depends upon your procedure. However you might just take some of the DNA and retransform it into a normal strain (DH5alpha or similar) and prepare plasmid from there to sequence and confirm.
Thank you for your answer. However, I tried to remove endo nucleasae using buffer in plasmid extraction kit. I alos used DH5a to obtins plamid. In both case i can not obtain consntration of plasmid.
If you are not getting good plasmid DNA from DH5a then it sounds there is a problem with the mini prep kit or a procedural problem. But if the strain is growing healthy with antibiotics in DH5a then there is no good reason you would not get plasmid DNA
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