Paired End Library Size Selection

[GeCKo]

Hello everybody,

we have problems with the final library size selction and the ampure beads. After the two cut-offs, we always get to small fragments. Did anybody tried to do the size selection by cutting it out of a gel instead of the beads? And maybe is it possible to do this at an earlier stage, when the carrier DNA is still in the sample, so you would actually see something on the gel?

Thanks a lot and good luck with your stuff.
Gecko

[sabrje]

Hi,

I have just sucessfully used the small fragment removal procedure from the rapid library protocol. This doesn't require 'calibration' of the ampure beads, but you just use the sizing solution instead. You have to adjust the volume of TE added according to the volume for your sample, but that's about it. Its an easier procedure in my mind.

Good luck

[Soulbee]

We have not yet tried using gel extraction but had a similar idea of using E-gels prior to the Library immobilization step to size select then continue on with the protocol and immobilize the beads after. Not sure if it would be better to gel size select before the library immobilization or to gel size select after the library amplification step, after getting rid of the magnetic beads.