I am about to perform a protein expression experiment and right now what I have is my gene in a pUC57 vector. As I'm not experienced in the procedure, I wanted to ensure I had backup DNA to work with (it's expensive!). For this reason, after digestion I am going to perform a PCR experiment on the linear DNA. My question is: is there any way to store the linear DNA product that I get from vector digestion, as well as the PCR product that I do not ligate into another vector and transform so as I can keep it as a backup? Would -20degC do?
Yes, you can keep DNA, whether plasmids or PCR products or purified digestion fragments, at -20oC. If I understand you correctly, you bought some pUC57 plasmid containing your gene, and you want to be sure not to lose it. The first thing you should have done is to transform a tiny bit of that plasmid into some competent bacteria. Then you can grow up one colony in a few mls of medium, cryopreserve a bit of bacteria at -80oC with glycerol, and extracted more plasmid from the remaining bacteria. It's always good to have your plasmid in two forms (free plasmid and inside bacteria) and in two different freezers (-20 for plasmid, -80 for bacteria) in case of freezer breakdown or human error. I hope you didn't digest all the plasmid you received and can still do this. If not, make sure you do it after you successfully subclone the gene into another vector.
Dear Brian,
You should ideally transform your pUC57 plasmid into DH5α cells and grow them up to 50ml. Miniprep the same so that the pUC57 plasmid stock solution doesn't remain a limiting source.
This way you wouldn't need to PCR since the miniprep elutions will serve the purpose and can be repeated when necessary. Agree with Shanshan on storage recommendations.
We always do that but I have noticed reduced ligation efficiency for fragments stored at negative 20 celcius. Therefore we store our fragments at negative 80 celcius.
Hi there,
Storing a plasmid or even a transformed cell is much more convinient. As mentioned, linear DNA products may be kept frozen but their stability is less than circular DNA.
All of these are good suggestions, especially the one about having a midi "seed" stock of your plasmid that you use only for transformation and propagation of more of the vector. All of the storage ideas are good too, but I think -80 degrees C for storage is a bit overkill, but may have been helpful for certain vectors.
That said, on several occasions over the years, I knew I was going to be subcloning several (5 - 20) viral genes into the same 2 or 3 expression vectors for DNA immunization. These were all for blunt-end ligations to keep reading the reading frames for fusion proteins intact, so I didn't have ssDNA sticky overhangs to worry about. I linearized a large amount of vector at the multi-cloning site, phosphatase treated it like crazy, and then gel purified the linear fragment away from the small proportion of uncut plasmid that was usually contaminating denatured, supercoiled vector that was resistant to digestion.
Once those vectors were quantified, I kept them at 4 degrees C and they gave high ligation efficiency and low background (vector alone ligation colonies) for years. I did the same procedure with sticky ended vectors, but probably didn't keep them at 4 degrees for as many years so I probably wouldn't have noticed much decrease in efficiency. The concentration of these stored vectors was usually 200 - 400 ng per uL in TE8 or 10 mM Tris pH 8. So, I'm from the camp that I avoid freeze-thaw cycles wherever possible.
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