I am using Gibson assembly method for insert my PCR product with the length of 3.6 KB into a vector, I excise the original vector to remove unwanted gene with specific restriction enzymes and use HIFI DNA ASSEMBLY for insertion PCR product into the vector, after assembly I used it for transformation finally miniprep, and for final approve of my work again use a enzyme that cut my plasmid exactly in the middle of the inserted gene, but the result on the gel is not satisfactory. what can I do?
Does your gene have the same sequence as the reference genome? Sometimes there can be SNPs which can affect restriction sites. When cloning from unconventional species I usually sub clone into a TOPO plasmid and have that sequenced so I am sure of what I am dealing with.
Did you run your PCR product on a gel to make sure you have the correct product? Did you run your digested vector on a gel to make sure the digestion worked?
Colony PCR is usually a better way of screening colonies for ones containing the correct insert, you can read more about it here: https://blog.addgene.org/plasmids-101-colony-pcr
Thanks a lot.
Yes I run both of insert and digested vector on the gel.
Colony PCR is a good way to approve the assembly.
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