Hello, basically I am quite new to PCR and would like some help. I have a vector that I am going to cut with XbaI and HindIII.
I want to insert a short signal peptide (15 residues, 45 nucleotides) that will retain both of the restriction sites (inserting other things after it later). I have a very rough sketch of an older project that, for the primers, included all 15 residues with the appropriate ends for the overhangs.
My question is, is the template DNA simply the vector that I want to insert in to as the primers would be able to anneal to the cut restriction sites or do I need to order a plasmid that has the signal peptide I want to amplify?
If my primers have 45 homologous nucleotides to each other, will that not create a huge Tm?
Thank you very much!
You don't need PCR for this step. You can simply denature the two (complementary) primers together and let slowly cool down. The primers will anneal to each other, similar to restriction fragment with the XbaI and HindIII sticky overhangs. Since the ends are not phosphorylated cloning efficiency will be low.
Not quite sure what you're asking, but typically for short sequences you can incorporate those into your primers. If it's more complicated than that I'd consider ordering a gBlock if there's not a clear way to get what you want with a combination of the primers, template, and vector.
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