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    • #8251
      piefke
      Participant

      hi!
      I´m trying to clone different 1,5 kb dna fragments into the pGemT-Vector and in a pQE-Vector. One time the cloning into the pGem worked. But after this success no ligation worked any more. I don´t know what is the problem.
      For cloning into the pGem I use a DNA polymerase that makes A overhangs.
      I checked the T4 ligase and it seems to be okay. And I tried different vector:insert ratios.
      To clone my fragments into the pQE Vektor I have to do a restriction first. I cut my vector and insert-dna with PaeI and SalI. Then I put it on a gel and elute my fragments. Then I do the ligation and make a religation-control too.
      Then I always find the same number of colonies on the ligation plate and the religation plate or even more colonies on the religation plate…..
      I really do not know what to change and what could be the problem. perhaps you have some ideas?! 😥

    • #76134
      weesper
      Participant

      Have you already picked colonies or just chucked the plates after comparing the number of colonies??

    • #76141
      piefke
      Participant

      yes. I picked colonies, but there were no inserts…. 🙁
      I checked this by pcr and restriction.

    • #76143
      SororSaudade
      Participant

      are you sure the restriction enzymes are working properly?

      I mean… If you’re digesting the fragments with 2 different enzymes you shouldn’t get any colony in the re-ligation control.
      Unless the ends are compatible… and then, your insert with that size could definitely self-ligate

      (hope I’m making myself clear…)

    • #76151
      blcr11
      Participant

      Sal I can be a very fussy enzyme. You might want to try and do your restriction digests sequentially if you can. Do the SphI (PraeI) digest first, then up the salt concentration (and add anything else Sal I likes, such as Mg or Mn or DTT, etc) and then add Sal I and continue the digestion for several hours to overnight–maybe add more than the minimum amount ot enzyme; or you can do a phenol extraction/etoh pptn and of the SphI digest and reconstitute in Sal I buffer and then continue on with the Sal digestion. You may have to reverse the order of digestion (Sal first, then SphI) if Sal has an absolute requirement for supercoiled DNA–I don’t remember if it does or not, but some enzymes don’t work so well on linear DNA. My guess is that you’re not getting complete digestion of one or the other enzyme, and I would lean toward betting on Sal I as the culprit.

    • #76161
      piefke
      Participant

      yes, I also think that the restriction could be the problem.
      I will follow your suggestion and try the restriction digest sequentially.
      I will report you about the results…. 🙂
      thanks so far….

    • #76553
      Duke
      Participant
      quote piefke:

      hi!
      I´m trying to clone different 1,5 kb dna fragments into the pGemT-Vector and in a pQE-Vector. One time the cloning into the pGem worked. But after this success no ligation worked any more. I don´t know what is the problem.
      For cloning into the pGem I use a DNA polymerase that makes A overhangs.
      I checked the T4 ligase and it seems to be okay. And I tried different vector:insert ratios.
      To clone my fragments into the pQE Vektor I have to do a restriction first. I cut my vector and insert-dna with PaeI and SalI. Then I put it on a gel and elute my fragments. Then I do the ligation and make a religation-control too.
      Then I always find the same number of colonies on the ligation plate and the religation plate or even more colonies on the religation plate…..
      I really do not know what to change and what could be the problem. perhaps you have some ideas?! 😥

      PaeI and SalI are pretty incompatible to each other in a single digestion step (check DoubleDigest on the Fermentas homepage)

      You need to sequentially digest with one enzyme, clean the fragment e.g. on a agarose gel and then igest with the other enzyme or use the Fermentas buffer for BamHI. But do not use more 10% enzyme in the reaction as high glycerol might inhibit enzyme action again.

    • #77542
      piefke
      Participant

      it worked. I think it was actually the restriction step that made problems.
      I sequentially digested and then the ligation and transformation were okay!!
      Thank you for your advices and ideas!!

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