homemade barcode adapters

[sasignor]

Hello,

My lab is working on developing homemade barcoded adapters, as in general the illumina kits are overpriced. No trouble coming up with the barcodes and adapter sequences, but as far as modifications go its hard to tell what is really necessary.

Some labs seem to use HPLC, while elsewhere desalting has been listed as being alright. This is a big one, as it adds a lot to the cost if you order many adapters. Any thoughts?

We were also planning on phosphorylating them ourselves, which should be reliable. Has any body else tried this? What about any of the other modifications?

For our nonbarcoded libraries we already use homemade adapters and primers and don't bother with most of the modifications and our results have been fine, but the investment in ordering all of these adapters is a bit higher I'd like to know others experiences.

Thanks!

[lance]

how's it going?

How are your efforts going with developing homemade barcoded adapters? I'm interested in pooling more than 12 samples per lane.

[sasignor]

Not so well, as it were. We have figured out that trying to phosphorylate them ourselves does not work, as we have started making out own non barcoded adapters as well. However, where those are working the barcoded adapters do not, and the libraries do not amplify. Other labs have successfully done this, so we aren't sure what our problem is. Good luck!

[eladSeq]

Any determination on whether the fusion primers need to be HPLC purified or just desalted?