I am working on re-designing 96 barcode adapters for RAD-seq (or GBS or genotyping-by-sequencing or ddRAD or whatever you prefer to call it) for our lab. I would think that adapters that would work for a PstI overhang would also work for an NsiI overhang, since the overhang is the same. However, I have noticed that the conventional wisdom is not to recreate the restriction cut site, which is why none of the standard barcode sets for PstI end in a C. By that logic, if I was going to use the barcodes for NsiI I would not want any barcodes to end in an A either. But if I heat-inactivate the NsiI before the ligation step, why does it matter if the ligation re-creates the cut site? Is there a bioinformatics problem, or is it just the wet lab problem of residual restriction enzyme un-doing the ligation?
Some people also like to digest again at a later stage to remove chimeric fragments that ligated together at the cut sites.
There is no problem with bioinformatics programs. They work with the information you give, no matter of recreated restriction sites.
You do it as a precaution to stabilize your construct and be sure they will be sequenced (or anything else you want to do with them). You never know if all the enzimes are inactivated or if there is some residues on your lab items.
Some people also like to digest again at a later stage to remove chimeric fragments that ligated together at the cut sites.
You're right - it probably doesn't matter if you are not doing another digest later. But if you design and buy barcodes, which can be expensive, probably better to design them this way in case your protocol changes in the future?
- Badges
- Science topic
- Similar topics
- Computer Science and Engineering
- Bioinformatics