Wednesday, February 3, 2010

Running our first PCR

Following our (stellar) success in running a mini-prep yesterday, Avital and I moved on to run a PCR on the plasmids we extracted.

If you do not know, the PCR (polymerase chain reaction) is a reaction in which we use DNA-polymerase, and enzyme that synthesizes DNA to be the reverse complement of a single strand DNA already in the mix. By moving between synthesis stages and "melting" stages where the DNA is separated into single strands, this reaction performs repeated synthesis of DNA. If we start with a single double strand, we get two after one round of amplification, four after two rounds, and so on, leading to an exponential increase in the number of copies.

The key to make a PCR reaction work is that the DNA polymerase has to have a small piece of double stranded DNA to start with (so it extends that piece). This allows us to control the reaction by introducing a sequence called a "primer" that hybridizes to a sequence it is complementary to. Once this happens, the DNA polymerase can perform its reaction. By using a pair of forward and reverse primers we can get synthesis on both strands.

The main work for us was taking the primers that we ordered and making them ready for use. This meant diluting them in very clean water (distilled, steralized, and then filtered) to a known concentration, and then preparing "work" tubes with lower concentration.

After, organizing all the primers (which took most of the morning), we moved to planing the actual reaction. This meant calculating metling temperature and designing the PCR mix.

(Avital calculating volumes in PCR mix)

Once all of the planning was done, the actual work was pretty quick. We created PCR mix, loaded the proper reaction components onto small PCR test tubes, and went to the neighbor's lab to run the reaction.


In setting up a PCR reaction, we essentially program a machince that can heat/cool the PCR tubes in a predefined schedule. The trick is that at high temperature (98C), the double stranded DNA "melt" into single strands. At lower temperature, the primers and the DNA template will anneal together. Finally, in an intermediate temperature of 72C  the special polymerase we are using (TAQ polymerase) is active and performs synthesis.


No comments: