I've been trying to digest my gene of interest and vector with BamHI-HF and NdeI in Cutsmart buffer. Both are known to have 100% efficiency in that buffer.
BamHI-HF is working fine but NdeI seems to be working less efficiently. What must be done to rectify the situation?
which target is not cutting. some enzymes like 2 cut sites to be present to cut well and many like additional bases either side of the recognition site to cut well so , for instance, if the vector is not cutting well then one possibility is that Bamhi is cutting quickly and well but if the sites are very close together then it may not be leaving enough spare bases to allow Nde to cut well. . In this case cutting with Nde1 first then BamH1 might work better but it is hard to say without knowing the sequence being cut. Possibly also the Nde1 may be old/degraded and just not working well. Does it cut well on its own do you know?
which target is not cutting. some enzymes like 2 cut sites to be present to cut well and many like additional bases either side of the recognition site to cut well so , for instance, if the vector is not cutting well then one possibility is that Bamhi is cutting quickly and well but if the sites are very close together then it may not be leaving enough spare bases to allow Nde to cut well. . In this case cutting with Nde1 first then BamH1 might work better but it is hard to say without knowing the sequence being cut. Possibly also the Nde1 may be old/degraded and just not working well. Does it cut well on its own do you know?
Thank you. Yes it does cut well on it's own. I'd set it up for single digestion as well to check i's efficiency.
I agree with Paul regarding NdeI inefficiency and degradation which need to be checked. You can try a higher concentration of NdeI in the digestion mixture and increase the incubation time.
To further illustrate Paul's point, compare here the efficiency of BamH I and Nde I when their target site is close to a DNA end: https://www.neb.com/~/media/NebUs/Files/Chart%20image/cleavage_olignucleotides_old.pdf
Out of curiosity, if both BamH I and Nde I work well on their own, how do you know it's Nde I that is not working?
Thank you all!
Hi Alejandro, I'd set up single digestion reactions for each of the enzymes using the same vector and incubated overnight. NdeI digested plasmid vector did get linearized. But I was facing problems while double digesting the plasmid with both the enzymes in a single reaction.
What vector did you use? How do you know that you are having problem with double digestion?
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