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  • #16
    Originally posted by Simone78 View Post
    Have you tried to do the assembly in solution as we describe in the paper? Just elute the tn5 from the column and add the oligos in equimolar conc with the Tn5 --> incubate 37 degrees --> ready to use. If that doesn´t work then you might have an issue with the Tn5.
    Best,
    Simone
    Hi Simone,
    I tried to produce the Tnp once again with the C3013 lately. But the strain seemed to reach plateau phase after the A600=1.3. No matter there is IPTG induction or not, same thing happened. However, according to your protocol, the A600 should reach 2.1 after IPTG induction for around 4h. The protein solution is still being dialyzed, I will test if there is any activity after that in any case. But the strain seems weird on our hands. Maybe you can give me some advices. Thanks!

    Cheers,
    Gary

    Comment


    • #17
      Hi all,
      I'm also trying to purify Tn5 using the pTXB1 plasmid, and I also don't have transpososome activity when assembled in solution with oligos. Does anyone have a slightly more detailed protocol (I'm a molecular biologist playing at protein biochemistry...) - especially regarding how to carry out the column steps, recommended equipment etc? I think my attempt was too drawn out in time (even though I did most of it in a cold room at 4C) and this may have led to incorrect folding/degradation. Any advice much appreciated!

      Comment


      • #18
        Originally posted by daniel007 View Post
        Hi all,
        I'm also trying to purify Tn5 using the pTXB1 plasmid, and I also don't have transpososome activity when assembled in solution with oligos. Does anyone have a slightly more detailed protocol (I'm a molecular biologist playing at protein biochemistry...) - especially regarding how to carry out the column steps, recommended equipment etc? I think my attempt was too drawn out in time (even though I did most of it in a cold room at 4C) and this may have led to incorrect folding/degradation. Any advice much appreciated!
        Up till now, I've already had seveal attempts. Nothing's wrong except for the activity. And now I am kinda puzzled. May I ask where did you get the pTXB1-Tn5 plasmid? Is it from Addgene?

        Comment


        • #19
          Originally posted by kobeho24 View Post
          Up till now, I've already had seveal attempts. Nothing's wrong except for the activity. And now I am kinda puzzled. May I ask where did you get the pTXB1-Tn5 plasmid? Is it from Addgene?
          I received the plasmid from Addgene, indeed. May I ask what equipment you use for the purification - i.e. AKTA etc? Have you tried both on-column and in solution assembly?

          Comment


          • #20
            Originally posted by daniel007 View Post
            I received the plasmid from Addgene, indeed. May I ask what equipment you use for the purification - i.e. AKTA etc? Have you tried both on-column and in solution assembly?
            I didn't use a specific equipment for protein purification. Bascially, I just followed every single step of the Genome Research paper. Moreover, I haven't tried on-column assembly yet, cuz I used to do the assembly in solution with a commersial Tn5 transposase, and it was not any issue.

            Comment


            • #21
              Originally posted by daniel007 View Post
              I received the plasmid from Addgene, indeed. May I ask what equipment you use for the purification - i.e. AKTA etc? Have you tried both on-column and in solution assembly?
              BTW, can you PM your email address to me? So that we can have more discussion about it.

              Comment


              • #22
                Originally posted by kobeho24 View Post
                I didn't use a specific equipment for protein purification. Bascially, I just followed every single step of the Genome Research paper. Moreover, I haven't tried on-column assembly yet, cuz I used to do the assembly in solution with a commersial Tn5 transposase, and it was not any issue.
                Did you find it difficult to achieve the onto-column flow rates reported in the Genome Research paper? Is there much difference between the paper and the protocol you received from Sandberg lab? I also tried without specific equipment, but hopefully this week or next will try again with the help of our protein core facility at my Institute.

                I'm not sure I have the ability to PM - apologies!
                Last edited by daniel007; 06-15-2015, 04:26 AM. Reason: Addition

                Comment


                • #23
                  Originally posted by daniel007 View Post
                  Did you find it difficult to achieve the onto-column flow rates reported in the Genome Research paper? Is there much difference between the paper and the protocol you received from Sandberg lab? I also tried without specific equipment, but hopefully this week or next will try again with the help of our protein core facility at my Institute.

                  I'm not sure I have the ability to PM - apologies!
                  Definitely, it is very hard to adjust the flow rates as the paper described. And the flow rate is not a critical parameter to the protein production if it is slow enough for ensuring the high efficient binding between intein-tag and chitin resin. Actually, there is no big difference between two protocols. And I assume the paper is more up to date. Looking forward to your positive result in the near future.

                  BTW, the reason why you can not PM should be you are still junior member, and I think there won't be such issue when you upgrade to member.

                  Comment


                  • #24
                    Hi all,

                    I am trying to go through the Tn5 purification as described in Simone's paper, but cannot seem to get the protein to elute from the chitin column. The small amount of protein that does come off is active, but after running a sample of the resin on a protein gel, most of the Tn5 seems to still be bound and uncleaved.
                    Have any of you had this issue? If not, how are you doing the elution?
                    I am currently eluting with 100mM DTT HEGX buffer, and incubating the column for 48h @ 4C.
                    Any help would be much appreciated!

                    Thanks a lot!
                    Emeric

                    Comment


                    • #25
                      Originally posted by Emeric Charles View Post
                      Hi all,

                      I am trying to go through the Tn5 purification as described in Simone's paper, but cannot seem to get the protein to elute from the chitin column. The small amount of protein that does come off is active, but after running a sample of the resin on a protein gel, most of the Tn5 seems to still be bound and uncleaved.
                      Have any of you had this issue? If not, how are you doing the elution?
                      I am currently eluting with 100mM DTT HEGX buffer, and incubating the column for 48h @ 4C.
                      Any help would be much appreciated!

                      Thanks a lot!
                      Emeric
                      I´m sorry but I can´t think of anything that you might do wrong! The paper describes exactly how we did it. As you also did, we add the DTT-HEGX on top of the column, drain out the buffer that was already inside the column, close it and, now that the DTT-HEGX is inside, leave it there over the weekend.
                      I just have one question: is your DTT fresh? DTT tends to oxidize easily. The self-cleavage of the intein from the CBD requires thiols and an old batch of DTT might be less efficient (although it is hard to believe that it has no activity at all, I admit).
                      For sensitive applications I always use single aliquots of "no-weigh DTT" (Pierce Biosciences) that comes as a powder and that I prepare at the moment and avoid freeze-thaw cycles.
                      /Simone

                      Comment


                      • #26
                        Hi Simone,

                        Thanks for your reply! I was using DTT that had been frozen, but never thawed until i needed it (it had only been frozen for a day or so). Maybe I got a bad batch? I will order some more, and not freeze it before use this time, hopefully that will help. I will also try using a bigger column to increase the surface area of resin to buffer. What size column do you use for your purification?

                        Thanks a lot,

                        Best
                        Emeric

                        Comment


                        • #27
                          Originally posted by Simone78 View Post
                          I´m sorry but I can´t think of anything that you might do wrong! The paper describes exactly how we did it. As you also did, we add the DTT-HEGX on top of the column, drain out the buffer that was already inside the column, close it and, now that the DTT-HEGX is inside, leave it there over the weekend.
                          I just have one question: is your DTT fresh? DTT tends to oxidize easily. The self-cleavage of the intein from the CBD requires thiols and an old batch of DTT might be less efficient (although it is hard to believe that it has no activity at all, I admit).
                          For sensitive applications I always use single aliquots of "no-weigh DTT" (Pierce Biosciences) that comes as a powder and that I prepare at the moment and avoid freeze-thaw cycles.
                          /Simone
                          It´s a regular 10 ml column that was included in the NEB kit (IMPACT). Used new ones and kept at +4 upon arrival.

                          Comment


                          • #28
                            Originally posted by daniel007 View Post
                            Did you find it difficult to achieve the onto-column flow rates reported in the Genome Research paper? Is there much difference between the paper and the protocol you received from Sandberg lab? I also tried without specific equipment, but hopefully this week or next will try again with the help of our protein core facility at my Institute.

                            I'm not sure I have the ability to PM - apologies!
                            Originally posted by kobeho24 View Post
                            Definitely, it is very hard to adjust the flow rates as the paper described. And the flow rate is not a critical parameter to the protein production if it is slow enough for ensuring the high efficient binding between intein-tag and chitin resin. Actually, there is no big difference between two protocols. And I assume the paper is more up to date. Looking forward to your positive result in the near future.

                            BTW, the reason why you can not PM should be you are still junior member, and I think there won't be such issue when you upgrade to member.
                            Originally posted by Emeric Charles View Post
                            Hi Simone,

                            Thanks for your reply! I was using DTT that had been frozen, but never thawed until i needed it (it had only been frozen for a day or so). Maybe I got a bad batch? I will order some more, and not freeze it before use this time, hopefully that will help. I will also try using a bigger column to increase the surface area of resin to buffer. What size column do you use for your purification?

                            Thanks a lot,

                            Best
                            Emeric
                            Sorry for quoting such an old thread but did any of you get the transposase to work? I am purifying it exactly as mentioned in the genome research paper but am facing the same problem. I get the correct sized protein on the gel but it has no activity. I have already tried the purification multiple times with no luck. If any of you got it working I would appreciate any tips on what you did differently.

                            Thanks!

                            Comment


                            • #29
                              Nextera XT and potassium acetate

                              Originally posted by Simone78 View Post
                              thanks! actually I just got some decent results today, just increasing the ionic strength of the final solution (adding potassium acetate). lower yield but similar profile at the Bioanalyzer
                              Hellohttp://seqanswers.com/forums/images/smilies/smile.gif, I am new to genomic practice but we want to test the Nextera XT protocol to obtain a genomic library of a plant (the reference genome is approximately 750Mb), and I have several basic doubts: in what step should I add the acetate of potassium? At what concentration was the potassium acetate solution? How many microliters did you use per sample? Thanks for your help!

                              Comment


                              • #30
                                Potassium acetate

                                Originally posted by Simone78 View Post
                                thanks! actually I just got some decent results today, just increasing the ionic strength of the final solution (adding potassium acetate). lower yield but similar profile at the Bioanalyzer
                                Please I am interisting in this modification for Nextera XT. Whay is exact protocolo for this?

                                Comment

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