Electrophoresis Lab Report
Abstract
Gel Electrophoresis is a method that separates molecules based on the rate of movement through the gel during the application of an electricity field. The direction the molecules move is based on the charge of the molecules because they will move towards their polar opposite. How fast these molecules move is also affected by the physical aspects of the molecule, such as size and shape, the density of the gel, and the strength of the electricity field. Because the density of the gel and the strength of the electricity field is the same for all of the molecules in a given electrophoresis chamber, the smallest particles will have the fastest rate of movement.
Introduction
In this lab, we were familiarized with gel electrophoresis and how it is used to separate the DNA fragments that result from a restriction endonuclease digest. The DNA loaded onto the gel is from lambda, a bacteriophage that was used in early studies of gene regulation. Restriction Endonuclease, also called restriction enzymes, are proteins that cleave DNA at specific sequences. Restriction enzymes were originally isolated from bacteria to protect the cell by cleaving the DNA of any invading virus. The bacteria’s own DNA is protected from cutting by being modified in a way that interferes with the restriction enzyme. Restriction enzymes find their cutting sizes by recognizing a short sequence of nucleotides called a recognition sequence. The recognition sequence may include the site where the restriction enzyme cuts or it may serve as the sequence the enzyme needs to recognize in order to cut a nearby sequence. Because DNA is negatively charged, an electrical field applied across the gel causes the DNA fragments in the samples to move from their origins (the wells) through the gel matrix toward the positive electrode. Smaller DNA fragments migrate faster than the larger ones, so restriction fragments of varying sizes become concentrated into distinct bands during electrophoresis.
Methods
Part I
First we casted a gel using a water bath, microwave, 6-well gel comb, masking tape, tray, and agarose gel. Using the masking tape, we covered the two ends of the gel casting tray to block out the agarose gel from falling out. Next we let the gel melt in the waterbath. Using protective gloves we took out the bottle, measured 15 mL of the liquid into a test tube and used the 15 mL in the casting tray. However right before we poured the gel onto the tray, we place the 6-well gel comb near the end of the tray. After allowing the gel to harden on the tray, remove one side’s masking tape. Before carefully sliding the gel without breaking into the chamber, pour buffer from a beaker into one side of the chamber. After placing the gel on the chamber add buffer until the level is approximately 2-3 mm above the top of the gel. Close the chamber and allow it to stay in that position for 24-48 hours.
Part II
Transfer the gel from the chamber back onto the plate so that it fits perfectly. Next, put the plate with the gel back into the chamber and pour the buffer into the chamber so that it is about 2-3 millimeters above the gel. Using a micropipette, take a color of dye and get 10 ul of it. Repeat this step 4 more times with different colors of dyes and using different micropipettes for each color. Next connect 5 alkaline batteries in a stack and connect the red cord and black cord from the batteries to their respective parts of the chamber. Once the battery pack is connected to the chamber, observe the appearance of bubbles and whether the different colored dyes are moving and if so in which direction of the chamber. After observation, disconnect the battery pack when the fastest moving dye sample is near the end of the gel.
Observation
We observed that we only had one band in our gel. This is because the lab is in part due to our not so high tech equipment and also because the lab has to be done in an almost perfect way. We did notice that when electricity was added to lab the liquids began to ‘bubble’. That is how we knew that it was working. We should have noticed different bands separated different distances for each of the three sample DNA’s.
Results
After recording our observations of the results that were provided to us we concluded the the EcoRi cut DNA strand was in fact the most mobile of the three. The HindIII cut DNA came in second and last was the uncut DNA. It makes sense that we got these results because it shows that EcoRi is splicing more DNA whereas HindIII is splicing less in comparison. The original DNA is not as mobile as the other two due to its greater length and more codons.
Discussion
1. We excluded the point plotted for the 27,491/23,130 bp doublet in the standard curve because the difference in distance was unrecognizable by the human eye. This, plus the fact that on this gel the fragments run as a doublet, tells us that the limitations of a 0.8% gel lies in the effectiveness of being able to separate fragments above a certain bp level.
2. If we want to separate DNA fragments that are 25,000 bp, 21,000 bp, 10,000 bp, and 6,000 bp then we need a percentage of about 0.5% or lower would be needed.
3. A gel percentage of 2.5% or lower would be needed to separate DNA fragments of 1,000 bp, 500 bp, and 100 bp.
4. To run the gel in 2 hours versus 1 hour, you can turn down the voltage by 50% (since you are doubling the amount of time) to 50V.
5.
6. The two fragments that made up the doublet would be the 1,080 bp and the 1,000 bp fragments because they are so numerically close together. To further separate the fragments, you could lower the percentage of the gel to create a bigger distinction between the close numbers.
7. A restriction enzyme is a protein that cleaves DNA at specific sequences.
8. If we were to cleave each restriction enzyme that shows up in the DNA strand, we would end up with 3 individual fragments.
9.