You have a few options and presence/size of the copied produces (which you can tell by agarose gel electrophoresis) can tell you different things: To answer this latter question, you can use PCR with cleverly designed primers. Each colony has lots of cells but they all have the same genetic makeupīUT this only tells you if the *plasmid* is inside the bacteria not if your gene is inside that plasmid. These bacteria grow and replicate to form individual “colonies” on a bacterial plate. Then I stick this RECOMBINANT plasmid into bacterial cells so the bacteria will make more of the DNA and/or protein.īut how do I know if the bacteria *really* have my gene in them? The plasmid vector has a selection marker – often an antibiotic resistance gene – so that if you grow the bacteria that should have it on food containing that antibiotic, only the bacteria that have the plasmid (and hence the resistance gene) are able to grow. There are different variations of PCR and reasons to use it, but I mainly use PCR during the process of MOLECULAR CLONING -> I copy an (edited) gene for a protein I want to study from one template and put that gene INSERT into a circular piece of DNA called a PLASMID VECTOR that has the “bells and whistles” I want, like “tags” to help with purification and start signals for turning the gene into protein. My templates come from someplace a bit different… Most frequently I use PCR to make lots of copies of a gene to put into a vector to put into bacteria to make more of the gene to put into other bacteria to make a bacmid to put into insect cells to make a baculovirus to infect more insect cells to make more baculovirus to infect more insect cells to express my protein. In this case, the template comes from cheek swabs, trace evidence, etc. You’ve likely heard of PCR being used to test for paternity or presence at a crime scene. more here: īut where does the template itself come from? That depends. We choose what region to copy by designing short pieces of DNA called PRIMERS to bookend the start & stop of this region (1 per strand) so that a protein called DNA POLYMERASE (DNA Pol) can copy each strand. Polymerase Chain Reaction (PCR) is a way to amplify (make lots of copies of) short stretches of DNA from longer pieces of double-stranded (ds) DNA we call the TEMPLATE. Note, this is an updated form of a past post – I’ve included more practical advice on analyzing sequencing data A technique called colony PCR can quickly tell me if my recipe *likely* got in there, but only sequencing can tell me if there are any typos. But before I try to get cells to make the protein, I want to make sure that the recipe got into the plasmid okay and there aren’t any typos. That plasmid serves as a vector or “vehicle” for getting (and keeping) the protein instructions in bacterial cells. Basically, there’s a me-altered version of the protein (aka a construct) which I want to express (get cells to make for me), so I took the genetic instructions for that protein and stuck that recipe into a circular piece of DNA called a plasmid. Cloning success! How do I know? Because my DNA sequencing data tells me so! I recently got back the sequencing results for a molecularly-cloned protein construct.