Paired end library, 3kb woes

[Dun]

Two questions, Miss:

The RNA trace you showed in other post, is it purified/sizing selected sample or straightly out from amplification reaction?

What is the amount of your final library?

THX a lot.

[MissDNA]

It is the purified/sizing slected library. I don´t run any chip or gel after the amplification reaction. As I told you before, I follow the protocol as is. The only modification I do is: after shrearing the DNA, I don´t do the ampure cleanup instead I concentrate my sample in a QIAquick column.

I use the concentration value and avg size I get from the Pico Chip, and throw in some excel spreedsheet I got from Roche years ago. That gives me the number of molec. and the values to make a 10e08 dilution to further dilute and use in the emPCR. Some of the libraries I have sequenced had barely 10e08 molec, so we made emPCR straight out of it and sequencing worked fine.

The 3kb PE library we sequenced yesterday yield great results, so great I am scared there is something wrong. It was a poor library. I am super curious to see the assembly.

[MissDNA]

Oh and as I mentioned before, when I see the peak after nebulization is too low, I proceed with 2 library amplication reaction and combine both in the last purification column step.

[ringo1230]

I have a question.
There is only the amount of 10ng DNA after reaction Plasmid-Safe ATP-Dependent DNase. I'm worried about it is that the experiment failed. Is it that the amount of DNA remaining everybody how much?