Paired end library, 3kb woes

[MissDNA]

Cool. Let me know how it goes.
The 2 libraries I prepared this week, titrated very well. Hopefully sequencing will be good.

[Coyk]

Well this gives me much hope! Thank you so much. I will take up the charge again and hope for the best.

[MissDNA]

I have prepared my fair share of 3kbPE libraries, and after circularization I NEVER saw a nice peak, as it shows in the manual, on the DNA 7500 chip. Most of the times I have an almost flat line. Still I continue with the protocol and most of the times I get a library because further on the protocol there is a library amplification step. Usually what I do is continue with 2 amplifications, instead of only one, and I combine the product of the 2 amplification in the final column purification step.
This week I prepared 2 libraries like that.
I really wonder if someone really sees nice peaks after circularization, and what can be done to improve circularization. Once per Roche´s tech support recommendation I increased the time of the circularization a bit. I went as far as 40 min only, but I am not sure if helped.

[Coyk]

Hello all,
I have made two attempts so far to make it through the paired end library protocol (Rev. June 2010 is the manual I've been referencing). I have made it through the circularization adaptor ligation, library span size selection, and fill-in reaction (up to section 3.5). After that there is a quantification step that I've done twice and both times gotten 22ng/ul which is more than enough to proceed. Apparently, though, I tend to lose everything by the time I run it on a 7500 chip after circularization and nebulization. The two most obvious possibilities to me are either the circularization adaptors aren't ligating properly or the circularization isn't happening and the subsequent DNase step chews my sample to bits. I was wondering if this has happened to anyone else and if there are any suggestions for troubleshooting.

Thanks!