Ibrahim's blog

Polymerase Chain Reaction (PCR), (week 7)

Figure 1  Agarose Gel electrophoresis
under a Gel analyzer software.

         Last week, DNA concentration was confirmed by the Nano-drop spectrophotometer. This week, our primary goal is to perform a PCR procedure. PCR is a technique where DNA is amplified by using specific primers for a particular piece of DNA. In short, PCR helps make copies of DNA fragments. The DNA will be amplified by the PCR, and the products of the reaction can be seen by the agarose gel electrophoresis (figure 1), where the gel is viewed under the UV light. Once in the UV light, it will be easy to determine the markers of the known sizes of base pairs, which helps us see the size of the DNA which has been amplified.

Gel electrophoresis

Figure 2 Gel electrophoresis

            Gel electrophoresis is a method used to isolate mixes of DNA, RNA, or proteins according to their molecular sizes (length in base pairs). The way it works is not that complicated. Now as we all know, DNA is negatively charged due to its phosphate group (figure 2, blue buffer). Since electrophoresis has positive electrodes, DNA will be forced to move towards the positive electrodes by an electrical field through the agarose gel matrix (figure 2). 

 As for our different types of plastics (LPDE/SPI 4, PVC/SPI 3…), we finally had them ground and up to go. But, before having to do anything with them, we had to sterilize them. Sterilizing means killing every living thing, which in our case, killing every little microbe in our plastic. The best equipment to do this job was using UV rays. UV rays can eliminate microorganisms at a specific wavelength ranging from 250 nm - 270 nm, but the only problem is that UV sterilizes the surface of our plastics (only the top part) and won't get the chance to penetrate all the way through the bottom of our plastics. Around two to three pinches of each plastic needed were transferred to a 100mL autoclaved beaker, and soaked by 100% isopropanol. Because the alcohol disinfects the plastic to kill any bacteria that may have been transferred to the surface during processing (since UV won't kill all the microbes found at the bottom of the plastics, only the surface). The beakers (figure 3) were left in the laboratory fume hoods so it won’t be contaminated, and wait for the alcohol to evaporate.

Figure 3 Beakers in laboratory fume hoods.
Notice the UV lights.