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文献和实验- normally blunt-end ligation of insert to vector does not reform the restriction site (therefore when you design the oligo that make sure it doesn't reform the restriction site when ligated), therefore only religated vector is digested. See one-tube PCR
), use a higher concentration of and ligase (~10 X). Higher concentration of ligase (~5x) is also useful for sticky end ligation if ligation time is short. d) Only use phosphatase when strictly necessary. Fewer steps means less problem
is dried and exposed to x-ray film, as discussed below for radiolabeled DNA sequencing. Protocol 1. Add the following reagents to a 0.5 ml microcentrifuge tube, in the order listed: sterile ddH2O q.s 10X kinase buffer
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