I have tried to separate the plasmids by extracting the DNA from agarose gel but I have only managed to make pRARE functional. I have been able to transform E. coli with pRARE that I obtained from this extraction but I do not obtain colonies when transforming with pET28a+. It should be noted that pET28a+ yields very little in this technique.
What extraction technique are you using to extract p28a+ ? A classical miniprep should work with a good yield for pET28a+. I you want to purify it from pRARE, just retransform the mixed miniprep into a strain that doesn't carry pRARE and select for kanamycin-resistant transformants. The odds of getting again a double transformant carrying both plasmids are very small, but you can check this by verifying that the transformants are sensitive to chloramphenicol, since this is the resistance marker carried by pRARE. You can then proceed to extract pure p28a+ from these transformants.
Here are a few strategies you could try to isolate the pET28a+ plasmid more effectively:
It's worth noting that each of these strategies has its own set of limitations and potential pitfalls, and it may be necessary to try more than one approach to successfully isolate the pET28a+ plasmid.
Pierre Béguin and Bertrand Cornu Thank you very much for the tips. They have been very useful.
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