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-   -   Nextera vs Nextera XT: whatīs the difference? (http://seqanswers.com/forums/showthread.php?t=31729)

Simone78 07-08-2013 03:36 AM

Nextera vs Nextera XT: whatīs the difference?
 
Hi all,
I tried to figure out the difference between Nextera and Nextera XT kit (for low input). With the Nextera kit I was never able to get good libraries when starting from <1 ng input DNA, while with the Nextera XT kit I got great results from <100 pg DNA.
I believe the enzyme is the same (I donīt think Illumina took the effort to engineer the transposase twice when they bought Epicentre Technologies...), so the difference must lie in the buffer.
I am quite sure that the Nextera kit uses a regular Tris+MgCl2 buffer, but what about the XT kit?
Any help is greatly appreciated!:)

luc 07-08-2013 10:23 AM

I would guess they reduced the enzyme concentration and added polyethyleneglycol (PEG) to the buffer to reduce the available volume (similar to the rapid ligation protocols).

Simone78 07-08-2013 01:32 PM

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

slees 10-02-2013 08:32 PM

I am also curious about this.
Also, why does Illumina suggest to use Nextera XT to make libraries only for small genomes? Can we directly use Nextera XT to make libraries for small amount (<1ng) of mouse/human genomic DNA instead of optimizing the buffer in Nextera kit?

kcchan 10-03-2013 10:00 AM

A diploid copy of the human genome is about 7pg. A 1ng sample would only have a bit over 100 copies of the genome, which is probably too little to generate a good library from.

slees 10-03-2013 10:05 AM

Good point. Thanks kccchan!

Simone78 10-03-2013 02:02 PM

for the genomic DNA 1 ng could be too little. I am doing single-cell RNA-seq and when starting from 1 ng or less (after RT & pre-amplification, of course) we get very high-quality data.

slees 10-03-2013 07:30 PM

Thanks Simone78. It will be great if you could also tell us something you have found about how the ionic strength of the final solution affects the reaction.

Simone78 10-04-2013 01:32 AM

Quote:

Originally Posted by slees (Post 117854)
Thanks Simone78. It will be great if you could also tell us something you have found about how the ionic strength of the final solution affects the reaction.

sorry, but that itīs going in a paper that we are going to publish (relatively) soon. Almost ready for submission, itīll still take a few months, I am afraid :)

slees 10-08-2013 09:54 AM

Thanks Simone78. Good luck with your paper! And would you please let me know when it is published?

slees 04-15-2015 10:44 AM

how to make good libraries from <1ng DNA using the Nextera kit
 
Hi Simone78, I was wondering if your paper has been published? I'm still interested in how to make good libraries from <1ng DNA using the Nextera kit. Thanks!

Quote:

Originally Posted by Simone78 (Post 117872)
sorry, but that itīs going in a paper that we are going to publish (relatively) soon. Almost ready for submission, itīll still take a few months, I am afraid :)


Simone78 04-15-2015 02:06 PM

Quote:

Originally Posted by slees (Post 170241)
Hi Simone78, I was wondering if your paper has been published? I'm still interested in how to make good libraries from <1ng DNA using the Nextera kit. Thanks!

yes, we published it in Genome Research last July. Hereīs the PMID: 25079858.
Best,
Simone

kobeho24 05-12-2015 12:40 AM

Quote:

Originally Posted by Simone78 (Post 170251)
yes, we published it in Genome Research last July. Hereīs the PMID: 25079858.
Best,
Simone

Hi Simone,
Recently I've got the plasmid from you guys as a generous gift, and gave a try to purify the Tn5 Tnp. After following every single step of the protocol (except for the competent strain C3013, we used BL21DE3 instead), there was no activity in the Tnp activity assay at all. The protein size is correct on the SDS PAGE Gel. So I guess there must be some issues in the transpososome assembly. Would you please give me some advice?

Best!

Simone78 05-12-2015 02:48 AM

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 doesnīt work then you might have an issue with the Tn5.
Best,
Simone

kobeho24 05-12-2015 03:12 AM

Quote:

Originally Posted by Simone78 (Post 172241)
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 doesnīt work then you might have an issue with the Tn5.
Best,
Simone

Actually, I did assemble the transpososome in solution @ 30C/RT for 1hr. Cuz I used to assemble a commercial unaasembled tn5 Tnp in this way. BTW, I was not only following the published paper but also the in-house protocol generously provided from your lab. So far, I might have to wait the arrival of the suggested strain, try one more time and see if it works. And, I was wondering that the incubation temperature after IPTG induction described on the Addgene webpage is 30C while your protocol shows that it should be 23C. As the temperature is crucial for polypeptide folding a correct way. I wanna make sure just in case. Thanks a lot!!!

Best!

kobeho24 06-09-2015 04:31 AM

Quote:

Originally Posted by Simone78 (Post 172241)
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 doesnīt 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

daniel007 06-13-2015 08:54 AM

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!

kobeho24 06-14-2015 06:33 AM

Quote:

Originally Posted by daniel007 (Post 175373)
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?

daniel007 06-14-2015 11:40 AM

Quote:

Originally Posted by kobeho24 (Post 175390)
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?

kobeho24 06-14-2015 07:14 PM

Quote:

Originally Posted by daniel007 (Post 175393)
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. :(


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