Paired-end reagents

[Anthony.287]

Hello all!
I'm preparing to embark on my first paired-end journey, and I have a few questions about what I need to have in stock. I'll be using the new protocol that came out for the Jr (March 2012), and it's clear about the Dynal beads and a few other reagents, but without having the paired-end kit in hand, I don't know what is in it!
Can anybody post the contents of the Paired-end kits? And if you know of anything else I may need to purchase separately, that would be great to know as well!
Thank you!!
Anthony

[Anthony.287]

Never mind about the kit contents; they are pretty easy to find at my454.com. Under "Documentation", click on "package inserts". There is a list of everything included.

[Rachael]

Hi Anthony, ordering the reagents can be quite tricky at the start because there are so many different enzymes and kits to buy all sold separately! How did your paired end library go with the new protocol?

[Anthony.287]

Rachael,
I think that I have all of the reagents, but I haven't had a chance to begin the process yet. I've been dealing with Rapid libraries for 4 weeks now, and I keep getting 0% enrichment. Once I get these done, I'll move on to the paired-end. Hopefully soon!

[Rachael]

Hi Anthony,

Have you tried FAM florometry or qPCR on your samples? Often that sheds some light on whether it is a sample issue or reagent issue.

We've just completed our first attempt with the new 3Kb paired end library protocol, the size is slightly smaller than expected (about 450bp average) rather than between the recommended 500 - 600bp, so I was wondering if anyone else who has attempted the the protocol had any troubles with the final size of the library.

Using the older 3Kb library protocol we had previously produced many beautiful libraries with great sequencing results. I think we will try making the library again with the new protocol to see if there's any improvement to the size.

I'd be very keen to find out how your PE library goes after making it. Good luck with your rapid libraries!

[Anthony.287]

Finally got those rapid libraries to work, and now I'm struggling with a few amplicon pools that keep failing to enrich. I'll let you know when I get to the PE library. It may be awhile!