Paired-End libraries with MID ?

[heleneb]

Dear all,

is it possible to pool some Paired-End libraries on the same PTP region ?

My gut feeling says YES, but I would like to be sure that I don't forget something...

Many thanks !

[Soulbee]

Technically, it should work... I thought this through as well and I don't see why it wouldn't. When i called Roche, they weren't quite sure and have not yet gotten back to me about it...

[flxlex]

Officially not supported, but technically not impossible. However, you are on your own (let us know if you succeed :-) )

[clarancer]

I always thought it theoretically possible to use the reagents from the rapid library construction kit after the fragmentation of the circularised DNA. If that is the case then it should simply be a case of using the Rapid MID adapters and they will then introduce the barcodes for you and allow you to multiplex your PELs. Because of the increased efficiency of adapter ligation at this stage adapter positive yields should be higher so may need less amplification and as a result redundancy should be reduced.

If I ever get round to trying it out I will let you know.

Clarancer

[cystron]

totally possible, done by some

Make your MID oligos according to the TCB for Titanium MIDs but omit the biotin. For the paired end protocol you do the ligation in the presence of the dynal m-270 beads and the affinity of the biotin is higher than the affinity blunt ended DNA has for one another. If you use biotinylated oligos you get no ligation and thus no PCR product a few steps later (in the PE protocol these PCR primers are biotinylated so you can later do the single strand isolation.


All that being said you will be on your own if it don't work.