plasmid DNA question

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    • #8045
      bluerose
      Participant

      Hello,
      I have an issue hoping if someone can answer it.

      I isolated and purified a plasmid with insert from Agrobacterium cells. Following protocol, I injected a small liquid culture of this into E. coli competent cells, heat shocked, and transformed onto agar medium. Transformed colonies grew. I isolated and purified plasmid DNA from these E. coli competent cells. Insert was indeed in this DNA as I checked on a restriction digest gel.

      I’d like to continue to use this same purfied plasmid DNA stock to perform DNA sequencing because the purification is extremely good However, I do not have enough DNA for the sequencing. My question is, how do I get more DNA from the purified plasmid DNA??

      I don’t know of any protocols to do this. Would I be able to take a small volume of my purified plasmid DNA, inject into E. coli competent cells, heat shock again, and retransform onto the agar medium?? Will this work???

      Any help is appreciated. Thanks.

    • #75015
      blcr11
      Participant

      If you’re asking, can you transform E coli with plasmid DNA you just preapared, the answer is, sure, that’s the standard method to propagate a plasmid in the lab. Plate out the transformants and pick colonies as you did before, grow it/them up and purify the DNA for sequencing, or for whatever else you have in mind. You can also make a glycerol stock from an overnight growth and store it at -70. Next time you might need the plasmid, you take a bit from the frozen stock, streak a fresh plate with the frozen bit of bacterial stock and next day you’ve got a fresh plate of colonies to work with again.

      If you’re asking whether there are protocols to amplify the copy number of plasmids during growth, the answer is, maybe. You can amplify some low-copy number plasmids by adding 170 micrograms/milliliter chloramphenicol. If I recall correctly, we would start the growth, wait unil the OD600 got to something like 0.7-1 and then add CAM and go overnight. (This was back in the days when if you needed a lot of DNA you had to do CsCl banding. I don’t miss those days and methods at all!) This trick doesn’t work with all bacterial strains–and it won’t work if your plasmid carries CAT. I’m pretty sure it works in DH5a and HB101, though the latter isn’t the greatest for plasmind preps.

    • #75016
      blcr11
      Participant

      Should also add, you don’t need to use very much of your plasmid prep to do the transformation. 0.5 microliter will probably do fine for a standard bacterial transformation, assuming your competent cells are in good shape.

    • #75023
      bluerose
      Participant

      Thanks for the reply!

      Apparently, people in the lab I work in don’t know this either because they said if I wanted more plasmid, I’d have to go all the way back and isolate the plasmid + insert from Agro cells again! But I have a great purified DNA stock here which I’d like to keep using if I can!

      I am using DH5a strain of E. coli cells, freshly made. Is it ok if the E. coli cells are already competent? Because my strain of E. coli cells are competent and stored at -70 to be used. I’ll just be taking the purified plasmid DNA and injecting, as you said, a small volume of it into these cells.

    • #75029
      blcr11
      Participant

      Usually, you make (or buy) competent cells in advance and store them at -70. You thaw a small aliquot on ice just before using. If your stored cells are competent, that should be all you need — well, that and your plasmid — and you should be in business. Surely your labmates were pulling you leg about about having to re-clone from scratch–at least I hope they were.

    • #75030
      bluerose
      Participant

      Well I wish they were. But no, they were not. Even the post doc here gave the same advice, to restreak from the original Agro stock and isolate and purify more plasmid DNA that way. But this wastes a lot of time and you may not get good insert in the end again even if you repeat with same techniques.

      Thanks for the protocol. I’ll pass it onto everyone.

    • #75036
      SororSaudade
      Participant

      I would transform E. coli with the plasmid + insert and then extract it with a kit, so that you can get high amounts of it very well purified. You can also store the transformed bacteria with glycerol at -70ÂșC and extract plasmid from them every time you want to.

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