Has anyone ever done this digestion. The digestion efficiency seems very low. The plasmid is very bright and the insert looks very faint on the gel? There is no dam methylation problem btw. Anyone know what the issue is?
My digestion times have been ~1.5 and ~2 hours. In the two hour case the inserts look brighter on the gel.
SpeI works best in it's own buffer. It may be more efficient to do subsequent digests in each enzyme's specific buffer.
They seem to be quite compatible. What buffer did you use? Did you add BSA?
Maybe one of the enzyme is out?
Did you try them separately on your plasmid?
They are both compatible with Buffer 4. I did not try them separately but will. Yeah I did add BSA but only to see if it made a difference and it did not. I normally dont add BSA to any digests and they all work fine.
I wouldn't expect any problem (and had none) using buffer 2 or buffer 4 and Biolab enzymes. However, careful with XbaI as overlapping dam methylation TCTAGAtc methylation prevents digestion. If you know the sequence, check it and regrow plasmid in dam-negative E. coli strain, if needed. Otherwise, one of your enzymes might simply be dead.
Ya, I faced this problem too. Both the restriction enzymes, XbaI and SpeI have the same sticky ends, CTAG. As a result, DNA cut by one enzyme can stick to DNA cut by the other enzyme. However, after ligation, the resulting sequence does not match the recognition site of either XbaI or SpeI. This is called a mixed site. The formation of mixed sites is the basis of BioBrick Standard Assembly where they can introduce new RE sites.
For cloning please don't use XbaI and SpeI together.
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