Restriction Mapping of Plasmid DNA

Purpose:
The purpose of this lab was to experiment with the usage of restriction enzymes on plasmid DNA using gel electrophoresis.

Introduction:
A restriction map is a visual of DNA with known restriction sites of that given sequence. A restriction map requires the use of restriction enzymes, which are enzymes that chop up a DNA molecule at a specific site. These DNA fragments are then separated by argos gel electrophoresis.  The distance between restriction enzymes can be found by comparing the patterns of the fragments. This process can be used to figure out information about the structure of an unknown piece of DNA. The fragments are compared to a lane of DNA markers which works a a size comparison chart. In this way the size of a DNA segment (number of base pairs) can be estimated.

Procedure: 

1) Carefully insert the buffer unto the gel's well by squeezing the pipette. Use the following table to make sure each buffer is in its correct well.

2) Bring the gel, which should look like the one below, to the electrophoresis area made by your teacher and place into the solution. 

3) The next day, take your gel out of the solution in the electrophoresis chamber and place it on a light box. It may help to mark the visible bands with some kind of marker to make them easier to see.

Discussion: 

PstI ad bands at 700 and 4700. PstI/ SstI had bands at 700, 2200, and 2500. PstI/ HpaI had bands at 700, 500, and 4200. PstI/ SstI/ HpaI had bands at 700, 1300, and 3000. Each digest added up to 5400 and had a band at 700. Because HpaI only has two fragments it must be circular. If the DNA were linear, there would be three different fragments with two different cites. There is only one PstI site because we know the HpaI has two cites already and there are only three fragments of DNA in the sample PstI/ HpaI. With circular DNA each site produces one fragment. We know SstI only has one site for the same reasoning mentioned before. Based on the position of the fragments on the gel, the 700 base pair fragment of HpaI is  unaffected by the two other enzymes. There is no fragment that appears only in the HpaI/PstI/SspI digest. This means that the  enzymes of HpaI, PstI, and SspI spliced the DNA in their respective locations/codes. Because  there are no other fragments present, HpaI, PstI, and SspI were the only restriction enzymes  used to digest this enzyme. 

Conclusion:

The purpose if this lab was to find out the number of cut sites present in the DNA sequence for each restriction enzyme and the position of the cuts relative to one another. We were successful in achieving a satisfactory result through the use careful observations and calculations. We were able to font the number and locations of all of the cut sites.