I have two different plasmids, 13.6kb and 10kb. Both the plasmids have SbfI recognition sites for sure. when I tried digesting both the plasmids with SbfI, only 10kb plasmid is getting digested. Most probable answer will be loss of restriction site but when I used a fresh vial of SbfI with higher fold I could able to see 40% digestion and its not star activity because I used it with other pair of enzyme which gave me right size of fragment. Also, the same 13.6kb plasmid is getting digested with other set of restriction enzymes without any smear. So my questions are
1. Whether size of the plasmid have any implication in activity of restriction enzyme-SbfI?
2. If plasmid size has any implication can I linearise the plasmid with any other restriction enzyme before digesting it with SbfI?
3. Based on my experience the smaller plasmid like pUC57 getting digested in 2hrs whereas bigger plasmid like pCAMBIA1305.1 takes overnight with SbfI. Has anyone faced similar issue.
Kindly let me know your suggestions.
I think that the shape of the plasmid has more effect on enzyme activity if the recognition site is hidden inside a folded structure it will cut slowly. On this scenario the buffer that the plasmid is dissolved in may also have an effect if it allows the formation of a stable conformation blocking the cut site. Sbfi is fussy about buffer conditions so there may be an effect if too much salt or the wrong pH exists as a result of the plasmid purification method
Have you tried different DNA stocks of the pCAMBIA plasmid? It seems to be the most likely explanation is some inhibitor in that plasmid preparation and a new DNA stock might not have that inhibitory factor. There should not be any notable effect of size on efficiency of digestion (and the difference between 10 and 13 kb is very minimal). It could also be context dependent as Paul Rutland suggests. But I would first rule out whether it is just a bad plasmid prep.
I believe the digestion is happening slowly due to the shape of the plasmid.
Kind regards
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