I have 5microgram DNA . I want to digest it with Nco1 and BglII enzyme thermoscientific through double digestion. I have not succeeded so far. Can anyone suggest optimal double digestion protocol using these two enzymes
Have you tried sequential digest, in their respective optimal buffers (Bufffer O for BglII and 1x Tango for NcoI)? Between digests, use a PCR cleanup spin column to remove salts.
Hi there,
Did you try the FastDigest versions of these enzyme, which means you can use them in a single step? The issue might be the proximity between the 2 sites, how close are they? If too close, sequential digest is definitely an option.
Please follow -
https://international.neb.com/protocols/2021/03/24/double-digest-protocol-with-standard-restriction-enzymes
If the digestion buffers are common and the restriction sites are separated by a minimum of 50bp, then you can try double digestion for at least 3 hrs. This works fine. Otherwise, you've no other option but digest separately.
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