PCR and Gel Electrophoresis

I used to teach a genetics lab and thought I would share what I taught my students. Before running polymerase chain reactions (or PCR), to make copies of your sample DNA, it is important to run a gel with your DNA extraction to see if the extraction worked. To run a gel, the DNA sample is loaded into wells on one side of a gel. An electrical current is used to separate out the DNA to analyze it. If your DNA extraction did not work it is a waste of money to run PCR on the sample. This gel is run in buffer TBE. TBE contains tris which raises the pH to 9, a boric acid which lowers the pH to optimizes it for the target enzymes, and EDTA which chelates metal ions and those stops DNeasy activity, which is the enzyme used for DNA extraction. The molecules in the gel separate based on charge, size, and shape. Personally, I like to use 1% agarose gel with TBE at 120 volts for 30 minutes.

gel

But what is agarose? It’s a seaweed polysaccharide that allows the gel to thicken. The more agarose you use the thicker the gel and the longer it will take your DNA sample to run through the gel. If you need to separate out really small fragments you would need a really thick gel but since we are looking to see how well the DNA extraction worked we can use a thin gel. You should also stain the DNA sample before putting the sample in the gel. If you don’t stain the sample you wouldn’t be able to see where your sample goes in the gel and run the risk if running your sample off of the gel. Gels also often have glyercide in them that binds to the DNA and makes it heavier so when you load your DNA into the well on the gel it is less likely that the DNA floats away. It another well a ladder is also typically used. In this case, we used a 1000 kb base pair ladder. A ladder is DNA fragments that are cut at known lengths. This allows it to act as a ruler for your other samples.

dna_agarose_gel_electrophor

The goal is to have bright bands through the gel. If there are no bands this could be because the DNA floated out when you loaded it into the gel or there was still ethanol in the sample from the extraction. IF the bands are smeared this means that the DNA is degraded as the loading dye can’t sit on it. Only DNA that produces bright bands should be used for PCR.

PCR basically replicates the DNA in a machine called a thermocycler after you add a number of reagents to your sample. It replicates at the 2n rate, where n is the number of cycles. Usually, PCR is run for 40 cycles. The sample DNA is double stranded so it has to be denature in order to replicate. 1st the thermocycler heats up to 94C to break the strands apart. In the 1st cycle, this step lasts longer, 3 minutes, to ensure that all the DNA is denatured. This mimics the actions of the enzyme helicase. Normally a cell would add primers at the point on the start region of a region that you want to replicate.

 

Thermocycler cycle
Made by Lauren Schramm.

 

We add these primers manually for this step called annealing. The primers are made from the genome of the organism you are working with. There is a lot that goes into picking the best primer so I am not going to go into much detail. The primers bond to a region we pick, and we use forward and reverse primers to capture the antiparallel nature of DNA. The next step is elongation in which copies of DNA are actually made. We need to add dNTPs, or synthetic base pairs to our sample, as the DNA needs bases to make more DNA. We also add something called taq polyerase, which is from the bacteria Thermus aquaticus which is a heat-loving bacteria. This is important because high temperatures of PCR denature other forms of polymerase.

taq
Taq polymerase

Finally, in the last step of the PCR cycle, the temperature drops down to 72C and the DNA closes up. In the premade Taq solution called go green Taq, there are also buffer for the primers, MGCL which helps the enzymes to bond easier, water to hold dNTPs, and dNTPs. Once all 40 cycles are complete, the last step holds the mix at 72C for 10 minutes to insure that the DNA is all closed up. THe mix is then held at 4C so that none of the enzymes can work.

For my lab, we used what is called a master mix. A master mix is a mix of everything you need for the individual reactions, except the sample DNA, in a large quantity. For example, if you need to do 16 reactions, you would multiple the amount of all reagents you need by 16 and mix all those together. In addition to speeding up the pace of the lab, this has another advantage, it is more accurate to pipette larger amounts.

 

PCR master mix
Made by Lauren Schramm.

 

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