Restriction Mapping of Plasmid DNA

The purpose of this lab was to use restriction enzymes in order to compare the DNA using gel electrophoresis.

Introduction: Restriction mapping of DNA allows sequences of DNA to be recognizable. Restriction enzymes cut the DNA sequence as well as allow you to find the position of the DNA. You can see how far the DNA travels using the method of gel electrophoresis.  How far the segments travel on the gel relates to how long the DNA fragment is. Smaller fragments of DNA move further than longer segments. Smaller segments have less DNA, and less to “carry” so they travel further. DNA has a negative charge and is placed on the negative side of the gel. A current running through allows the DNA to travel to the positive side of the gel. Smaller bands are on the positive side of the gel, while larger pieces stay by the negative side of the gel. You can tell which fragments are smaller, larger, and which may have been been broken off from their original fragment.  

First, we got a pre made gel that had already been set and placed it into a plastic holder. This allowed us to determine which was the positive and negative end.

Next, we loaded the gel. In each well of the gel we placed the different DNA. We used 1 pipet per tube of DNA in order to extract the DNA and place it into the appropriate well without contamination. We made sure to place all of the DNA inside of the well so that it took up the entire space.

Then, the electrophoresis chamber was closed and power was run through it. After the current was applied for some time, the gel started to move from the wells to the other end of the gel. After left for some time, we removed the gels and were able to see the bands that had formed due to the current running through the gel.

    After we removed the gel from the electrophoresis chamber, we used a light box in order to examine where the DNA bands had formed.

Using a bag we marked where each of the bands had been and where the wells were. This allowed us to have a permanent source of data incase something were to happen to our gel or bands so that we could not see where they ended up.

We were then able to measure the bands and find out what the distances were from one band from the next.    

Discussion: We were able to use gel electrophoresis in order to map a plasmid. There are two PstI restriction enzyme cut sites since the channel in he fell shows two markings. There are two SspI sites since the channel cut SspI creates two new fragments from one large fragment cut by PstI. There are two HpaI restriction enzyme cut sites. Since the HpaI creates new two fragments from one large fragment cut by PstI. When the DNA was cut with HpaI,HpaI/SspI, and HpaI/PstI the fragment length of 1093 was made. This means that HpaI cuts the DNA at this point and that it will remain unchanged when oh her restoration enzymes are added to HpaI. The 4700 segment cut by HpaI is cut even more when you add SspI or PstI. When you add SspI the segment is cut into pieces 1986 and 1700 long . When you add PstI the 4700 segment is cut into pieces 2140 and 514 long. Based on these observations we were able to label our plasmid to see which restriction enzymes cut where. Based on our plasmid pictured above, we decided that HPAI cuts both at 514 and 805 are further cut by SSPI which cut at 1159 and PSTI at 1900.We compared this data to our gel to see how many bands each had and compared them to the row before to determine where the next restriction enzyme would cut. We know the largest cut is PSTI because of the distance that it travels and because the other restriction enzymes do not cut that piece of DNA, it is only cut into about half which is still a large piece.

Conclusion: From restriction mapping, we are able to determine where certain restriction enzymes cut. This helps us determine the size of the fragments of DNA. From gel electrophoresis, we are able to compare a test piece of DNA to other fragments of DNA being tested. Also, on the gel by the distance that the DNA fragment travels we are able to tell the relative size. Based on the distance traveled on the gel and the other restriction enzymes we are able to draw a plasmid (pictured above) and determine the cuts in the plasmid for the relative sizes of DNA.