[Simone78]

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

[Simone78]

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

[Chalk]

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.

[Simone78]

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

[json]

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

The relatively high concentrations of Triton-X 100 could be interfering with the light scattering... as long as the enzyme is active, then thats really all that matters. Simone, I'm curious if you ever tested eluting the Tn5 at lower detergent concentrations, we've found that the high concentration of detergent, coupled to the fact that it can't be dialyzed away and also tends to get concentrated during Amicon concentration makes it difficult to determine the protein concentration accurately... again, I realize that activity is the metric that matters at the end of the day. Do you have a sense as to whether there is a critical Triton concentration that is required to keep the transposase soluble?