Lab 9 PCR and Gel Electrophoresis

Introduction

The purpose of these labs was to use the polymerase chain reaction (PCR) technique to multiply the isolated DNA from the previous lab 8.  This will allow us to visualize DNA using agarose gel electrophoresis and some colored dye. In order to multiple DNA segments we have to use the PCR technique. This technique generates a large amount of copies DNA, generally of one from something you need for a test using initially a very small sample. For PCR to replicate DNA like the typical natural process, we must supply all of the components normally present for replication in that of a test tube. So we have to force polymerase to synthesize new DNA strands that we need, what can go wrong?! (zombies?). Just add some heat and walla, so we need a thermocycler that will increase the temperature to roughly 95 degrees Celsius. This now denatures the cell allowing us to add whatever we need.

Gel electrophoresis is performed once PCR is completed. This helps us see whats going on. Gel electrophoresis is the technique of separating fragments of DNA based on their size. DNA is injected into a well in an agarose and a dye is used. This will brighten up the data and now will show us the different sizes of DNA present.

Methods and Materials

Obviously, we have to grab those important supplies and fight over what color pipette we want to use. After we are ready to go grab a 25 uL PCR tube following the addition of 2.5 uL 10X Standard Taq Reaction Buffer, 0.5 uL 10 mM dNTPs, 0.5 uL 10 uM forward primer, 0.5 uL 10 uM reverse primer, 2 uL Template DNA, 0.125 uL Taq DNA polymerase, 18.875 uL Nuclease-free water. Now we need some heat so throw it on a thermocycler that will reach 94 degrees Celsius for the next 5 minutes. This will cause a initial denaturation of template DNA.

Now we run 45 cycles of our sample at 94 degrees Celsius for 30 seconds, then 50 degrees Celsius for 30 seconds, 72 degrees Celsius for 30 seconds. Finally, when you’re tired of writing the word Celsius, we need the thermocycler to sit at 72 degrees Celsius for 7 minutes. This will cause changes in temperature that will now allow for Taq Polymerase to copy template DNA strands at an exponential rate. Placed in the freezer at 4 degrees Celsius until it is needed for further experiments.

Now for electrophoresis, we need some squishy agarose gel that is premade by the professor or an assistant. Put gel tray in the gel box, make sure its sealed. Place gel comb where wells are made in the gel for your sample to be placed into. The comb and agar is placed into the electrophoresis. Now once gel is solidified, place 8 uL of ones DNA sample that should be mixed with 2uL of 5X loading dye. Note: Do not pour too much into the gel tray or you will not form clean wells upon solidification!

 This dye is used so that the DNA is visible as it mixes across the agarose. The 10-uL sample is then transferred into a well in the agarose. Mix with the pipette. Now look at the wells. The wells are located by the negative terminal and are completely submerged in a salt solution to allow electricity to flow efficiently through all of the agar. This leads to the power supply which happens to be connected to about 100 volts. Given that the DNA is negatively charged it is attracted to the positive terminal and begins to move toward the other side. The power is turned off when the dye front is near the end of the gel. The distance that the DNA migrates depends on the size of the DNA in question. The larger the strand, the slower it will travel. To get the data, we need to remove the gel and place on a transilluminator. We can take that picture now (selfie).

Results

No results but a picture of what it should look like.

Discussion

The DNA ladder is used to identify the size of the DNA strand, thus also telling us the length of the DNA. A restriction enzyme cuts DNA at a specific location so we can find what DNA will be synthesized for PCR. We can find the location DNA mainly in the nucleus or even the mitochondria which in return form chromosomes. This is how we fit such long strands in such a tight package. Different bands on the gel correspond to different lengths of fragmented DNA strands. The longer the DNA fragments, the slower they will travel through the agar shown above. If multiples show, then we know that the restriction enzyme cut the DNA in multiple places.