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Old 03-31-2010, 09:30 AM   #1
nickloman
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Default New England Biolabs Reagents

I hear that New England Biolabs are offering 454 reagents. Has anyone had any experience with them? Which parts of the Roche workflow do they replace? They are supposed to be keenly priced.

Any tips appreciated
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Old 03-31-2010, 09:35 AM   #2
nickloman
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Just realised there's an advert at the top of the page.

Details here:
http://www.neb.com/nebecomm/tech_ref...es/NEBNext.asp

Looks like this is a replacement for the library preparation stage (i.e. fragmentation
and ligation of sequencing adaptors/MIDs) but you still have to buy the
emPCR kits and sequencing kits/PTP from Roche?
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Old 04-07-2010, 12:47 PM   #3
AKroxy
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the kits don't include adaptors.

This is ok for the standard library, since you can get the adaptor sequences,
but if you want to use the rapid library adaptors, you have to buy the rapid library prep kit from Roche
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Old 04-08-2010, 10:56 PM   #4
RCJK
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Would it work if you just used the RL MID adaptors kit as your adaptors?
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Old 04-08-2010, 11:22 PM   #5
AKroxy
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Wish I'd thought of that!
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Old 04-09-2010, 04:31 AM   #6
pmiguel
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Quote:
Originally Posted by nickloman View Post
Just realised there's an advert at the top of the page.

Details here:
http://www.neb.com/nebecomm/tech_ref...es/NEBNext.asp

Looks like this is a replacement for the library preparation stage (i.e. fragmentation
and ligation of sequencing adaptors/MIDs) but you still have to buy the
emPCR kits and sequencing kits/PTP from Roche?
That link doesn't work for me. Here is another one:

http://www.neb.com/nebecomm/tech_ref...xt/NEBNext.asp

I think the critical replacement here is an enzyme, "ds Fragmentase" for the nebulizer. Much easier to process large numbers of samples that way.

Also always worth crediting NEB with providing open protocols that specify the composition of reagents they provide. The current standard -- where companies somehow get away with providing reagents for scientific research without providing this information (claiming that it is "proprietary") is obviously detrimental to the progress of science. Why do we put up with it?

--
Phillip
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Old 04-09-2010, 09:05 AM   #7
nickloman
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Ah OK, I guess that would be somewhat helpful. We've got the rep visiting next week so I'll report back on what they say.

Just seen this advert too which looks interesting for 454 library quantification:
http://www.kapabiosystems.com/produc...ary-quant-kits
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Old 04-12-2010, 07:30 AM   #8
gogreen
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Hi Nickloman, I use the NEBnext sample prep kit for the Illumina library prep. This works quite well for me and Its much cheaper than the Illumina reagents. I don't know about the Roche sample prep kit, but if the performance of illumina kit was something to go with, I believe it should be good for Roche too! Try to get a trial kit from them.
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Old 02-14-2011, 08:08 AM   #9
mchotalia
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Hi,

Has anyone had problems using the NEBnext sample prep kit with Paired-end adapters? I recently made libraries with genomic DNA and observed a strange band at 120-150bp..this isn't primer/dimers as they run much lower nor the library. I do not do a size selection before the enrichment but after.

I tried to TA clone the band and observed lots of positive colonies and few negative ones. However, when i look at the results only a 5bp product has ligated (very weird).

Any ideas would be much appreciated.

Thanks
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Old 02-14-2011, 09:23 AM   #10
ChickSeq
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In order to use the Rapid Library Adaptors, use: NEBNext® Quick DNA Sample Prep Reagent Set 2, CN: E6080L or E6080S.

For standard Titanium Adaptors, use: NEBNext® DNA Sample Prep Reagent Set 2, CN: E6020L or E6020S.

The only difference is that the Quick DNA prep kit contains the additional polymerase that will add the extra base onto your library to allow the Rapid Library Adaptors to ligate properly.

Sidenote: We have been using these reagents as replacements for the 454 reagents on several non-standard shotgun library prep processes for over a year and have had no complaints regarding performance. I highly recommend evaluating these in your lab because of the potential cost savings.

Also, someone mentioned the KAPA biosciences quantitation. We also use this method and have gotten extremely good results, even with libraries that were unable to be detected on an Agilent Bioanalyzer chip. It's a little more time consuming to set up (because of the dilutions and then the data analysis on the back end), but it's really worth it to recover low quantity libraries post-library prep.
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Old 02-21-2011, 03:17 AM   #11
vtosha
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Quote:
Originally Posted by mchotalia View Post
Hi,

Has anyone had problems using the NEBnext sample prep kit with Paired-end adapters? I recently made libraries with genomic DNA and observed a strange band at 120-150bp..this isn't primer/dimers as they run much lower nor the library. I do not do a size selection before the enrichment but after.

Thanks
We got this band too, if we had not performed a size selection before enrichment.
I think it is adapter's dimers (if I not wright I will be glad to know a truth). They can contaminate qPCR results, so now we perform size selection before enrichment each time.

Last edited by vtosha; 02-21-2011 at 03:30 AM.
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