Restriction Mapping Tutorial

This is a restriction mapping tutorial designed to teach the process of determining the restriction map of a clone that has been isolated in pBluescript.

The Problem:

You have an unknown 3.0 kb piece of DNA in the plasmid pBluescript (the plasmid is from Stratagene). You need to prepare a restriction map of the cDNA insert to set up your future experiments. This is what you know about the plasmid before you start:

Figure 1

The insert is cloned into the Eco RI site of the polylinker, which cuts the Lac Z gene in half. You have this restriction map of pBluescript, so you know what enzyme sites are on the extreme right and left of the insert and how big pBluescript is (3.0 Kb). These are the only places these sites appear in the vector plasmid.

Your first strategy is to cut the plasmid with all of these polylinker enzymes to see which of them are in the insert and where the sites are relative to the site in the vector. You set up restriction digestion for each of the enzymes and allow them to cut for one hour. After the incubation period, you load them on a gel. You run the digestions from the left side on one gel and the right side another gel.

Take each gel to the dark room and look at it under the transilluminator.

Left Side Enzymes
Right Side Enzymes

Interpretation:

To interpret the results, take a piece of paper and write the name of each enzyme and the sizes of the bands that they produce under one of the following headings:

No Sites         Single Cutters          Multiple Sites




When estimating the sizes of the bands, remember that the sizes of fragments larger than the 2.3 Kb marker are hard to estimate and therefore inaccurate. Use only the estimates of the smaller bands and make sure that the sum of all the bands equals the total plasmid size (6.0 Kb).


Mapping Single Cutters:

The enzymes that fall in the first category are already mapped - they don't exist in your insert. The digestion cut the plasmid only once, in the polylinker, and it was linearized.

The enzymes that fall in the second category have one site inside your insert; two bands resulted from one cut in the polylinker and one inside the insert of the circular plasmid. The size of the smaller band is the distance from that enzyme's site in the polylinker to its site in the insert. The larger band is the part of the insert left on the vector after the digestion plus the vector plasmid (3.0 Kb). For example, if enzyme X is on the right side and gives a 2.0 Kb band and a 4.0 Kb band, then the site for X is 2.0 Kb from the right side. If enzyme Y is on the left side and gives a 0.5 Kb band and a 5.5 Kb band, then the site for Y is 0.5 Kb from the left side:

Figure 2



Draw a line on the bottom of your paper and make a restriction map of the single cutters. Be sure to note which side of the insert the enzyme is on in the polylinker. It will make a big difference in where the sites map out.

After you have done this (but not until) click here to see a partial restriction map and check your work.

When you have successfully mapped the single cutting enzymes, go on to map the multiple cutters.



Mapping Multiple Cutters:

If you were successful at that part, congratulate yourself but don't get too cocky. That was the easy part. Now you have to map the enzymes that cut two times inside the insert. This is done by cutting the plasmid with two enzymes at once - with the double cutter and one enzyme that has only one site in the insert.

The fact that you have three bands on the gel means that the enzyme cuts in three places. One is in the polylinker, which is part of the vector plasmid, so it's position is known - either on the left side of the insert or the right.

The position of the second site is easy to find. The vector plasmid is 3.0 Kb. The largest fragment is larger than 3.0 Kb. Since there are no other sites for this enzyme in the vector, the largest fragment must contain the vector plus some of the insert. (Look at the origin of the largest fragments in figure 2.) Therefore, the second site is "the size of the large fragment minus 3.0" kilobases from the opposite side of the polylinker. That is, if the multiple cutter is on the left and you have a 4.3 Kb fragment, then the second site is 1.3 kb from the right side.

So far you've only used the info given with the vector (figure 1) and the size of one of the fragments and you've mapped two of the three sites. Now you will use the other two fragments to find the position of the third site.

The third site is the most difficult. With these first two sites mapped, there are two possible positions for that third site that will give the same two remaining fragments. On a piece of paper, draw two circles like in figure 2 and determine what the two possibilities are. Then take the position of one of the single cutters that you mapped previously and map it on these two circles. Determine the sizes of the four fragments that you would get in either case. There should be two possibilities (unless the single cutter cuts inside the largest fragment i.e., the 4.3 kb in this example). Note especially which of the two fragments bands gets cut by the single cutter. This band will disappear from the double digestion.

When you've decided how to tell the difference between the two possibilities, go to the selection inputs below and see which of these results is what you get. From this you can determine the placement of the third site.

Hints:

Choose two of these enzymes:

After you have chosen a single cutter and a double cutter, the results of the digest.

When you have learned which of the two possibilities is correct, add the position of this enzyme into your partial map. The complete the restriction map will show the positions of each of the enzymes in the insert.

When you have finished your restriction map, check your work by looking at the complete restriction map.




Conclusion:

Note how much information you now have on the insert. You can isolate any piece of DNA that you want from the clone by choosing one or two enzymes, digesting the plasmid, running a gel, and then cutting the fragment out of the gel. This fragment can then be cloned back into a vector plasmid that has been cut with the same enzyme(s).




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