Electrophoresis Lab
1. Abstract
The process of electrophoresis is used by many different types of scientists. We reproduced it by using a gel we hardened, a buffer to keep it from becoming too stale, and also electricity. The second day we worked we put a dye on the gel so that it could be easier to see where the DNA had travelled. We examined the movement of DNA through the gel, and where the different distances at which the pairs separated. We recorded the distance travelled and also the relative mobility was recorded.
2. Introduction/Background Information
This lab covered the topic of electrophoresis, which is a process used by forensic scientists to solve criminal cases. It is also used for paternity tests and to diagnose genetic diseases. DNA electrophoresis can be used to map the order of restriction enzymes within chromosomes. RNA electrophoresis is vital in gene expression. In this specific lab, DNA electrophoresis was studied. The DNA used in the lab was obtained from bacteriophage lambda, a virus. There were 3 DNA samples, one uncut, one incubated with Hindlll and EcoRl.
3. Method/Procedure
We conducted our experiment at New Tech High School. Our group observed the travel of bacteria through a gel, to examine electrophoresis. We did this experiment on three different types of DNA per group, each group having similar DNA. We started the experiment by place the gel into a tray with a special comb in it so the gel could have holes. We then put the DNA into the gel using a micropipet. Next we put the tray with the gel and DNA into a box and filled it with buffer just until it was slightly over the tray. The box had 75 volts of electricity put into it to make the DNA travel.
After we took the tray with the gel out of the box we put a dye on top of it so that over night the bands don’t disappear. We recorded the distance the DNA travelled in the gel, along with the Rf (relative mobility) of the DNA. The base pairs were already given to us.
4. Data
5. Discussion
The DNA bands that appear at either ends of the gel spectrum, when the restriction enzyme HinDIII is used, are known to be a doublet. The large pieces of DNA move with less separation and therefore appear as one, when two strands are present. The 0.8% gel used in this experiment does not allow larger strands of DNA to move more rapidly and therefore cannot be accurate without prior knowledge and corrective calculations.
Using a gel closer to 1.4-1.8% would yield a band separation that would allow for distinguishing large strands more effectively.
For measuring smaller DNA fragments, a gel around 3.5-4.0% would slow down the movement of the DNA, and therefore allow more precise measurements.
In a situation where one must leave the gel active for two hours, lowering the voltage will slow down the movement through the gel and prevent runoff.
DNA is naturally a negative particle, this means that to run this experiment you should align the DNA on the negative side, so that it can be drawn towards the positive when current is active. If the DNA moves the wrong direction, it means that the tray was put in with the DNA nearest to the positive end.
{ - well / // / + } {1,400 1,080-1,000 400} Running the gel for longer would allow for more separation between the 1,080-1,000 doublet.
A restriction enzyme is a protein specifically designed to split DNA at precise Base-Pair locations.
5’-AAAGTCGCTGG/AATTCAC-3’ -------- 3’-TTTCAGCGACCTTAA/GTG-5’
Using a 0.8% gel and 100v, I would use three different enzymes on each tube of DNA and inject them accordingly in the wells of the gel. After running the current through the gel for one hour I would stop it, dye the gel, and the measure the band separation. Then I would plot this information and compare that to the known BP statistics for the tubes ambiguously.
6. Conclusion
Gel electrophoresis is a procedure that separates DNA, RNA or proteins through the rate of their movement through a gel that is under the influence of an electrical field. The gel is loaded with wells that are inserted with DNA. The direction of movement is controlled by the charge of the molecules and since DNA is a negatively charged molecule, because of the phosphate groups in its backbone, it moves towards the positive pole. The rate of movement is controlled by the size, shape, and density of gel, as well as the strength of the electrical field. DNA is cut by restriction enzymes, which are highly specific and cut DNA within precise recognition sites, which are palindromic. Ligase is the enzyme that joins molecules. RFLP stands for restriction fragment length polymorphism.
