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You make up this sample in little tubes (at right, from www.ga-international.com), and run the temperature program in an instrument called a thermocycler (at right, from www.uni-saarland.de). All this device does is heat and cool your sample, but it does so very precisely according to a program that you write into the instrument. Typically this program runs for around 2hrs. At the end of it, you will hopefully have a tube full of linear double stranded DNA corresponding to the region of the template flanked by the two oligonucleotide sequences.



For the purposes of cloning, there are two tricks about PCR we take advantage of frequently. First the oligos don't have to exactly match the template. They have to be pretty darn close, but a few mutations here and there won't screw it up. However, whatever sequence is present in the oligos ends up in the final pcr product. The result of this is that you can change the sequence of the DNA at the ends of a pcr product by careful design of your oligos. This process is generally called "site-directed mutagenesis," and we'll be using it later in the tutorial. The other important trick is that you can "pin" additional sequences to the 5' ends of your oligos without screwing up the PCR. Those sequences similarly will end up on the ends of your pcr product. Frequently, we'll pin the recognition sequences for restriction enzymes to the 5' end of oligos so that we can cut the DNA and then ligate it into a cloning vector.

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