Gel Electophoresis Lab

Background Information:

Gel Electrophoresis is a type of DNA technology that's used to separate nucleic acids or proteins that differ in size, charge, or other properties. The separation of the molecules depends on the rate of their movement in a polymeric gel in an electrical field. The distance traveled by a DNA molecule is inverse to its size. In other words, if the DNA molecule is larger in size, then it will travel a shorter distance because it has more resistance due to its size. On the contrary, smaller DNA molecule have less resistance so they travel faster down the gel. The DNA is always placed on the cathode side (negative) because DNA is also negative so when the power source is added the  the DNA will repel away from the cathode and towards the anode(positive) side. Restriction fragments can be used to cut up the DNA molecule into bands.
Purpose:
The purpose of this lab was to use restriction enzymes to sequence DNA. Using single, double, and triple digests, we tried to figure out the number of cut sights present in the DNA sequence for each enzyme as well as the position of those cuts relative to one another.

First our teacher cast an agarose gel with wells included.

Using a pipet we loaded the contents of a reaction tube (DNA with restriction enzymes) into a well in the gel.

We repeated this procedure with each reaction liquid into a different well.

When we finished loading it looked like this.

We put our gel in the electrophoresis chamber, and allowed the DNA to electrophorese until the bromphenol blue band was about 2cm from the end of the gel.

We removed the gel.

And we examined it on a light box. We assigned sizes to the lambda DNA size marker bands, then approximate sizes to the unknown DNA fragments, and determined the total size of digested DNA.

Data and Graphs

This shows the marker's lengths, so that we can
estimate the lengths of the DNA strands for the rest of the lanes.

Discussion: 

 Gel electrophoresis was used to determine the size of the unknown DNA sequence. The size of sequence was determined to be  about 5500 base pairs in length by comparing against a known lambda DNA sequence. When the unknown DNA sample was cut with restriction enzyme PstI, there were two bands at approximately 750 and 4700 base pairs.  The PstI had cut the unknown sequence twice. PstI and SspI there were three bands at approximately  750, 2140, and 2700 base pairs. This shows that the SspI sequence had only cut once. PstI and HpaI there are two bands at approximately 750 and 4700 base pairs.  Since there were only two bands, this shows that adding the HpaI did not cut the sequence. PstI, SspI and HpaI there are three bands at approximately 750, 2858 and 1093  base pairs. This is expected after since PstI cut twice, SspI cut once, and Hpal did not cut at all; giving a total of three cuts and three bands on the gel. Further investigations could look into the discrepancy between the bands in the lane containing PstI and SspI and the lane containing PstI, SspI, and Hpal.

Conclusion: Our results demonstrate how different restriction enzymes cut the plasmid into different sizes. All the different wells should have DNA fragments of the same size because we used the same DNA, but our variables were the restriction enzymes that digested the DNA at different locations. PstI only cut the plasmid into 2. PstI and SspI combined created a plasmid with 3 different sections. PstI and HpaI also created 3 different sections even if they one of them wasn't clearly visible on the gel. The last well with all the enzymes shows a combination of the fragments.