insertion of short sequence - (Jul/24/2006 )

Hello,

I would like to know if there is an easy way to introduce short segment of nts (15) into a plasmid.
I have a plasmid and I want to introduce a linker peptide between two fused fragments !!!

any idea


thanks

PCR mutagenesis?

U could PCR amplify one of the fused fragments with the extra 15 bases or u could make a linker for 15 bases and clone it in.

thanks for your ideas

Actually, my fragments were fused together and cloned into a pcDNA vector without adding the linker between them. Now, I want to insert short segement of nonplar a.a. between the fragments. I don't have restriction site between the fragments.

I'm thinking of using mutagenesis but I don't know how it works with insersion of more than one nucelotide.

anyone did that before!!!!!!!!!!!

i can suggest you to design 2 primers with appropriate restriction sites and pcr the vector. i've attached a picture for better explanation.

Thank you guys,

what about using two primers having the short sequence (let's say divided between them 7 nt on each) to amplify the whole plasmid using high fedility polymerase. Dpn digest the parental plasmid and self ligate the linearized PCR product (primers are phosphorylated) and transform cells after that.

anybody tried that before???

I would suggest overlap PCR: make 2 primers (REV and FWD) that carry the sequence you want to introduce and use them with corresponding "wild-type" primers - do both PCRs separately, purify the products and use some 40 ng of each product together as the new template: they will anneal and serve themselves as primers and template at the same time.

I used this technique for the introduction or loxP sites into a targeting vector - it worked surprisingly well. Hope the attached scheme is of help.