i have plan to add a secretion signal to my plasmid. the point is here that i do not have any source to PCR that sequence and at last do GA. i need to synthesize by myself. i want to know is it possible to use one long pair of plasmids with overlap at the 5' end and do PCR whole plasmid. after that transfer to E.coli and get the circular plasmid? the size of whole plasmid is 3.5kb. Thanks in advance for your response
What you could do is split the sequence as 35bp tails between the forwards and reverse primer (no overlap needed). Amplify the vector at the insertion point then use a DNA kinase to phosphorylate the DNA end (or order phosphorylated primers). Do a blunt ended ligation to seal the plasmid then transform it into ecoli.
Adron Ung yes, that is what I mean. considering that the plasmid is very small this should be suitable.
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