At some point in a cloning project it will be necessary to construct a restriction map of a plasmid. This is a rapid way to organize and confirming the structure of a vector. This will involve mapping restriction digest fragments into a circular map.

There is a systematic approach to constructing a map, which is illustrated in the sample problem below. Once you have mastered the sample problem (plasmid pRIT450), complete at least pRIT451 (Question 1) and your personal question (Question 1).

Working with actual restriction fragments from a gel is a bit messier than these problems because of experimental error in determining fragment sizes. Sometimes the mathematical approach used here needs to be supplemented with some common sense.

The circular plasmid pRIT450 has two restriction sites each for the restriction enzymes PvuII, EcoRV, and BtrI. From the data below, determine the size and restriction map of the resulting plasmid. The fragment sizes are given in kilobasepairs (kb):

Enzymes        fragments (kb)

PstI           7.0   4.0
EcoRI          6.0   5.0
BamHI          8.9   2.1
PstI + EcoRI   4.3   3.3   2.7   0.7
PstI + BamHI   6.1   2.8   1.2   0.9
EcoRI + BamHI  5.0   2.1   2.1   1.8

The first task is to determine the size of the plasmid. We can observe that all fragment length for each enzyme or enzyme combination is sums to 11 kb in the table above.

Since all digestions give the same total sum, we can say with some confidence that the total size is 11 Kb and that there are probably no distinct fragments with the same size in the digestion with a single enzyme.

It is important to remember that there is not necessarily only one solution, but the suggested solution has to fit the observed fragment sizes.

The first step is to select a reference digestion. It does not matter which one. In this example we choose PstI. I think that it is easier to work with a linear representation of the plasmid. The figure below represent the plasmid more or less to scale with the fragments formed from PstI. I chose to start by locating the largest PstI fragment (7) at 12 o'clock followed by the 4 Kb fragment (below).

The second step is to locate two enzymes on the restriction map. It does not matter which ones. We start by locating PstI and EcoRI together on the map: t

In the first attempt, we distributed the fragments by size (empty boxes). This organization is obviously incorrect. We can recognize that the 7 Kb fragment can only be made up of 2.7 Kb and 4.3 Kb and the 4 Kb fragment of 0.7 Kb and 3.3 Kb. The (1) and (2) combinations fits the observations.

We now do the same thing with the PstI and BamHI combination.

We can see that the 7.0 PstI fragment could only be made from the 6.1 and 0.9 fragments and that 1.2 and 2.8 fits the 4 Kb fragment (above). Both (1) and (2) fits the observations so far.

The final challenge is to combine the two double digestions.

We know that EcoRI + BamHI gives 5.0 + 2.1 + 2.1 + 1.8. We can identify a 5.0 fragment in the middle of the PstI + EcoRI (1) digestion. This fragment is preserved in the EcoRI + BamHI digestion. We can see that this only happens in the PstI + BamHI (2) digestion.

We now list the fragments by the order they appear (PstI + EcoRI + BamHI) (above).

Question 1:

This is an individual question for each student. Go to the Google Spreadsheet for TP12. You should find your name in the leftmost column. In cell F2, there is a link to a google document that has a table of fragment sizes immediately below your name.

Your task is to use your data and solve the restriction map. Formulate you restriction map with enzyme names and fragment sizes as for the first example student "Max Maximus".

It does not matter which enzyme you chose to begin your map.

You can solve one of more of the examples below if you feel that you need more practice.

Plasmid pRIT451 was cut with SmaI, BglII, and AvaI. From the data below, determine the map.

SmaI         5.9   4.3	
BglII        8.2   2.0	
AvaI         5.3   4.9	
SmaI + BglII 5.4   2.8   1.5   0.5	
SmaI + AvaI  3.3   2.6   2.3   2.0	
AvaI + BglII 5.3   2.1   2.0   0.8

Plasmid pRIT452 was cut with PstI, HindIII, and EcoRI. From the data below, determine the map.

PstI            6.8   5.9  	
HindIII         6.5   6.2	
EcoRI           9.2   3.5	
PstI + HindIII  4.8   4.2   2.0   1.7	
PstI + EcoRI    5.4   3.8   3.0   0.5	
EcoRI + HindIII 6.2   3.5   1.8   1.2

Plasmid pRIT453 was cut with SmaI, HindIII, and EcoRI. From the data below, determine the map.

EcoRI           7.7   1.6
HindIII         7.4   1.9
SmaI            6.6   2.7
EcoRI + HindIII 4.5   1.9   1.6   1.3
EcoRI + SmaI    5.7   2.0   0.9   0.7
SmaI + HindIII  2.7   2.5   2.2   1.9

Plasmid pRIT454 was cut with PstI, HindIII, and EcoRI. From the data below, determine the map.

PstI            6.0  5.3
HindIII         5.8  5.5
EcoRI           6.5  3.0  1.8
PstI + HindIII  4.0  3.8  2.0  1.5
PstI + EcoRI    3.5  3.0  2.5  1.8  0.5
EcoRI + HindIII 5.0  3.0  1.5  1.0  0.8

Plasmid pRIT455 was cut with BtrI, HindIII, and EcoRI. From the data below, determine the map.

EcoRI           8.5 2.0 0.5
HindIII         5.6 5.4
BamHI           6.5 4.5
EcoRI + BamHI   4.5 2.2 2.0 1.8 0.5
EcoRI + HindIII 4.3 4.2 1.2 0.8 0.5
BamHI + HindIII 3.4 3.1 2.5 2.0

Plasmid pRIT456 was cut with PstI, HindIII, and EcoRI. From the data below, determine the map.

EcoRI           5.8 3.3 2.9
PstI            7.4 4.6
HindIII         6.8 5.2
PstI + EcoRI    3.3 3.3 2.5 2.1 0.8
PstI + HindIII  6.3 4.1 1.1 0.5
EcoRI + HindIII 3.6 3.3 2.2 1.6 1.3

Plasmid pRIT457 was cut with BamHI, HindIII, and PstI. From the data below, determine the map.

BamHI           5.1  4.5  3.8	
HindIII         8.1  5.3	
PstI            13.4	
BamHI + HindIII 5.1  3.3  2.5  2.0  0.5	
BamHI + PstI    5.1  4.5  2.5  1.3	
HindIII + PstI  8.1  3.3  2.0