One question you might be asked is the general procedure required to run PCR. In this case, make sure you understand these steps:
https://biology-forums.com/index.php?action=gallery;sa=view;id=48610Here's an animation explaining the polymerase chain reaction:
https://videos.homeworkclinic.com/watch/659/polymerase-chain-reaction-pcr-animation/Let's begin with
figure 1.
On the left-most column, we have know marker sizes, we use this column to help us determine the sizes of the DNA that has been amplified. Now look at the 3rd column. We see a dark line at around 100, and few more above it. That means we have DNA pieces in the sample that have been cut to those sizes. The second column is a control, to show the reader that without the proper preparation, no DNA will be amplified.
In
figure 2, we notice two more columns tagged along to our original picture. The two columns show an increase in annealing temperature, leading to fewer bands appearing. Why? Because increasing the annealing temperature in PCR enhances the specificity of primer binding to the target sequence, thereby reducing
non-specific amplification from unintended primer binding. However, excessively high temperatures can compromise amplification efficiency too, so don't over do it.
In
figure 3, we see a third factor being studied, which is the concentration of magnesium chloride changes from lane to lane. Increasing the concentration of MgCl2 in PCR can have and impact on amplification. Magnesium ions (Mg2+) are essential cofactors for the DNA polymerase enzyme, facilitating primer annealing and DNA strand extension. So increasing MgCl2 concentration within a certain range can enhance PCR efficiency by promoting specific primer-template binding and in turn improving DNA synthesis. However, too much MgCl2 concentrations can lead to non-specific amplification, so this needs to be optimized MgCl2 to achieve the best results.
