SEQanswers

Go Back   SEQanswers > Applications Forums > Sample Prep / Library Generation



Similar Threads
Thread Thread Starter Forum Replies Last Post
Nextera XT Normalisation JoeChris38 Illumina/Solexa 5 05-26-2014 02:58 AM
Nextera XT gillt Sample Prep / Library Generation 0 03-06-2013 05:05 PM
Nextera kits HMorrison 454 Pyrosequencing 3 11-01-2011 01:00 PM
Problems with Nextera John.Sawyer Sample Prep / Library Generation 3 04-11-2011 07:33 AM
nextera niceday Sample Prep / Library Generation 5 09-10-2010 11:31 AM

Reply
 
Thread Tools
Old 07-08-2013, 03:36 AM   #1
Simone78
Senior Member
 
Location: Sweden

Join Date: Oct 2010
Posts: 173
Default 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!
Simone78 is offline   Reply With Quote
Old 07-08-2013, 10:23 AM   #2
luc
Senior Member
 
Location: US

Join Date: Dec 2010
Posts: 271
Default

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).
luc is offline   Reply With Quote
Old 07-08-2013, 01:32 PM   #3
Simone78
Senior Member
 
Location: Sweden

Join Date: Oct 2010
Posts: 173
Default

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
Simone78 is offline   Reply With Quote
Old 10-02-2013, 08:32 PM   #4
slees
Member
 
Location: NC

Join Date: May 2012
Posts: 11
Thumbs up

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?
slees is offline   Reply With Quote
Old 10-03-2013, 10:00 AM   #5
kcchan
Senior Member
 
Location: USA

Join Date: Jul 2012
Posts: 172
Default

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.
kcchan is offline   Reply With Quote
Old 10-03-2013, 10:05 AM   #6
slees
Member
 
Location: NC

Join Date: May 2012
Posts: 11
Default

Good point. Thanks kccchan!
slees is offline   Reply With Quote
Old 10-03-2013, 02:02 PM   #7
Simone78
Senior Member
 
Location: Sweden

Join Date: Oct 2010
Posts: 173
Default

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.
Simone78 is offline   Reply With Quote
Old 10-03-2013, 07:30 PM   #8
slees
Member
 
Location: NC

Join Date: May 2012
Posts: 11
Default

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.
slees is offline   Reply With Quote
Old 10-04-2013, 01:32 AM   #9
Simone78
Senior Member
 
Location: Sweden

Join Date: Oct 2010
Posts: 173
Default

Quote:
Originally Posted by slees View Post
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
Simone78 is offline   Reply With Quote
Old 10-08-2013, 09:54 AM   #10
slees
Member
 
Location: NC

Join Date: May 2012
Posts: 11
Thumbs up

Thanks Simone78. Good luck with your paper! And would you please let me know when it is published?
slees is offline   Reply With Quote
Old 04-15-2015, 10:44 AM   #11
slees
Member
 
Location: NC

Join Date: May 2012
Posts: 11
Default 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 View Post
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 is offline   Reply With Quote
Old 04-15-2015, 02:06 PM   #12
Simone78
Senior Member
 
Location: Sweden

Join Date: Oct 2010
Posts: 173
Default

Quote:
Originally Posted by slees View Post
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
Simone78 is offline   Reply With Quote
Old 05-12-2015, 12:40 AM   #13
kobeho24
Member
 
Location: HKUST, Hong Kong

Join Date: Apr 2015
Posts: 32
Smile

Quote:
Originally Posted by Simone78 View Post
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!
kobeho24 is offline   Reply With Quote
Old 05-12-2015, 02:48 AM   #14
Simone78
Senior Member
 
Location: Sweden

Join Date: Oct 2010
Posts: 173
Default

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
Simone78 is offline   Reply With Quote
Old 05-12-2015, 03:12 AM   #15
kobeho24
Member
 
Location: HKUST, Hong Kong

Join Date: Apr 2015
Posts: 32
Smile

Quote:
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 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 is offline   Reply With Quote
Old 06-09-2015, 04:31 AM   #16
kobeho24
Member
 
Location: HKUST, Hong Kong

Join Date: Apr 2015
Posts: 32
Smile

Quote:
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 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
kobeho24 is offline   Reply With Quote
Old 06-13-2015, 08:54 AM   #17
daniel007
Junior Member
 
Location: London, UK

Join Date: Feb 2015
Posts: 5
Default

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!
daniel007 is offline   Reply With Quote
Old 06-14-2015, 06:33 AM   #18
kobeho24
Member
 
Location: HKUST, Hong Kong

Join Date: Apr 2015
Posts: 32
Default

Quote:
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?
kobeho24 is offline   Reply With Quote
Old 06-14-2015, 11:40 AM   #19
daniel007
Junior Member
 
Location: London, UK

Join Date: Feb 2015
Posts: 5
Default

Quote:
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?
daniel007 is offline   Reply With Quote
Old 06-14-2015, 07:14 PM   #20
kobeho24
Member
 
Location: HKUST, Hong Kong

Join Date: Apr 2015
Posts: 32
Default

Quote:
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.
kobeho24 is offline   Reply With Quote
Reply

Tags
buffer, nextera, nextera xt, tagmentation

Thread Tools

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off




All times are GMT -8. The time now is 11:07 PM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2017, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO