My colleague has come up an idea of using two inserts to ligate into one vector at the same time, which is like : vector-SpeI-Insert A-BamHI-Insert B-NotI-vector
(Spel-Insert A-BamHI, BamHI-Insert B- Notl, Vector digested with Spel and Notl).
Then he transformed the system into E.coli and today the colonies showed up.
Unfortunately, the verification PCR only shows the band of Insert B fragment.
(vector is around 3kbp, Insert B is around 1.8kbp, Insert A is around 700kbp, the verification PCR's predicted size is around 2.5kbp containing the part of Inert A and B) We are confused about the result and I really wish to hear from your suggestions.
How many colonies showed up? Likely there is an issue with the digestion of insert A. It's possible for insert B to function alone at low frequency in this set up, but if you're getting many colonies then something else is going on.
Your design should work so I would recommend rigorously checking that all your digestions are functioning correctly, and then you're cleaning up the digestions so no enzyme remains.
For inserting multiple inserts at once, Gibson assembly is much more effective than traditional cloning with restriction enzymes. You can read about Gibson assembly here, it's pretty cheap and very efficient: https://blog.addgene.org/plasmids-101-gibson-assembly
Thank you so much for your reply.
We have realized that using restriction enzymes to invert two or more inverts have low efficiency so we are preparing to adopt Gibson assembly.
Thank you so much!!
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