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    • #15582
      biology_06er
      Participant

      Hi there,

      So I recently ligated my gene into a Pet32a vector using restriction sites using EcoR1 and Xho1. From another Pet vector I have to create primers for the GFP insert containing BgII and EcoR1 sites. Just wondering, once I amplify my GFP (using BgII/EcoR1 sites I assume), how do I ligate it into my original construct..Do I cut my original constuct with EcoR1 and BgII (because looking at the vector map, Pet32a has a BgII site and ligate?)

      Cheers,
      b_06er

    • #107142
      JackBean
      Participant

      you should thing about this before you start your work, because you should be sure, that e.g. BgII won’t cut in your gene of interest or that it doesn’t cut your vector more times. But yes, I would expect that.

    • #107194
      daniel.kurz
      Participant
      quote JackBean:

      you should thing about this before you start your work, because you should be sure, that e.g. BgII won’t cut in your gene of interest or that it doesn’t cut your vector more times. But yes, I would expect that.

      I agree with Jack Bean. If you’re not careful, you will end up with your gene of interest in the wrong spot. My experience is that so long that your cut doesn’t degrade the end and become blunt ended and doesn’t get a strange mutation it should ligate in nicely. Best of luck.

    • #107216
      biology_06er
      Participant

      Hi there,

      Thanks for the replies…I had a relook and it seems my GFP insert already has BgII and EcroR1 inserted (as it would as it is already in the Pet vector) then i wouldnt need primers right?… I could cut the GFP insert straight from the Pet vector using BgII and EcoR1 and likewise cut my original construct with those 2 restriction enzymes and ligate?

    • #107217
      JackBean
      Participant

      wait a moment, so you have a gene, which you want to clone into pET vector and GFP, which you have in a pET vector? So why do you want to cut the GFP?

    • #107221
      biology_06er
      Participant

      Hi,

      I have 2 pet vectors…one containing GFP and one containing my insert…..the GFP was has a bgii and ecor1 site and my insert has a xho1/ecor1 site….the plan is to fuse my insert with GFP so im thinking if the gfp is already in a pet vector, i need to cut it out and insert it into the vector contiaing my insert?

    • #107279
      JackBean
      Participant

      OK, I see. Then you should be able to just cut the GFP out of the vector, but remember, that with PCR you increase the number of copies, with restriction you will get at best the same number of GFP copies as you have vectors.

    • #107405
      daniel.kurz
      Participant

      Agreed.

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