Tweet
I need to shear long-range PCR product in order to produce biotinylated baits for enrichment (in-solution capture). In the past, I've used a Covaris for shearing but now I don't have one available. I do, however, have many restriction enzymes at my disposal. But I have no experience with digests, especially for this purpose.
The products are between 8-10kb each, spanning the entire mt genome. In Webcutter I see that AluI and HaeIII are blunt end cutters and are each *supposedly* cutting enough to produce fragments under 1kb, which is what I need. But when we run the digests out on a gel, no matter how much enzyme or how long we incubate, large products remain.
Has anyone successfully enzymatically sheared DNA for this purpose- with efficient results of the capture?
By having blunt-end cutting enzymes, can I skip the blunt-ending PCR that occurs before ligation of my biotinylated adapters? (I'd like to reduce the number of purification steps, given how much material is lost in a MinElute column, for example).
Any insight is appreciated!
The products are between 8-10kb each, spanning the entire mt genome. In Webcutter I see that AluI and HaeIII are blunt end cutters and are each *supposedly* cutting enough to produce fragments under 1kb, which is what I need. But when we run the digests out on a gel, no matter how much enzyme or how long we incubate, large products remain.
Has anyone successfully enzymatically sheared DNA for this purpose- with efficient results of the capture?
By having blunt-end cutting enzymes, can I skip the blunt-ending PCR that occurs before ligation of my biotinylated adapters? (I'd like to reduce the number of purification steps, given how much material is lost in a MinElute column, for example).
Any insight is appreciated!