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Old 06-14-2015, 09:08 PM   #21
kobeho24
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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.
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Old 06-15-2015, 04:18 AM   #22
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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 at 04:26 AM. Reason: Addition
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Old 06-15-2015, 10:50 PM   #23
kobeho24
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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.
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Old 07-06-2015, 03:36 PM   #24
Emeric Charles
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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
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Old 07-07-2015, 05:27 AM   #25
Simone78
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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
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Old 07-07-2015, 09:41 AM   #26
Emeric Charles
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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
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Old 07-07-2015, 01:29 PM   #27
Simone78
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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.
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Old 10-11-2016, 06:19 PM   #28
tn5
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Quote:
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!
Quote:
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.
Quote:
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!
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Old 12-09-2016, 10:23 AM   #29
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Question Nextera XT and potassium acetate

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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!
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Old 12-14-2016, 05:53 PM   #30
hydnazul
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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?
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Old 12-30-2016, 01:22 AM   #31
Simone78
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Originally Posted by hydnazul View Post
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!
Hi,
potassium acetate was/is used just to replace the Tris-HCl as tagmentation buffer and it´s used at the same conc (10 mM for the 1x). After trying many different tagmentation buffers we noticed that there is no dramatic difference between TAPS, Tris-HCl, KOAc and HEPES, as long as the conc is 10 mM, the pH around 7.5-8.5 and you have 5 mM MgCl2 (1x).
Best,
Simone
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Old 12-30-2016, 01:27 AM   #32
Simone78
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Originally Posted by hydnazul View Post
Please I am interisting in this modification for Nextera XT. Whay is exact protocolo for this?
I refer you to our detailed protocol published in:
Picelli et al., Genome Research 2014, "Tn5 transposase and tagmentation procedures for massively scaled sequencing projects.".
Let me know if you need additional info or have specific questions!
Best,
Simone
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Old 04-18-2017, 03:24 PM   #33
Chalk
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Hi Simone,

Have you ever noticed a tendency for the Tn5 transposase to aggregate after elution from the chitin resin? The protein looks like the correct size on denaturing SDS-PAGE but when I look at it through SEC-MALS for molecular weight, the protein appears to be severely aggregated.
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Old 04-20-2017, 05:25 AM   #34
Simone78
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Originally Posted by Chalk View Post
Hi Simone,

Have you ever noticed a tendency for the Tn5 transposase to aggregate after elution from the chitin resin? The protein looks like the correct size on denaturing SDS-PAGE but when I look at it through SEC-MALS for molecular weight, the protein appears to be severely aggregated.
Hi,
we never checked MW with SEC_MALS, just by regular gel. In general if the protein was eluted then it was functional. In the beginning we were losing it in the earlier steps (in inclusion bodies) but if that would happen you wouldn´t see it coming off the column. I guess you have tried to do the tagmentation and it didn´t work, right?
Sorry, right now I don´t have a better idea of what might be wrong!
/Simone
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