Lambda GT10: Titre of a Phage Stock

Introduction:

Lambda GT10 is a genetically engineered version of the wild type lambda virus. It has been changed in two important ways in order to make it more useful for molecular biological manipulations. First, a large piece of unnecessary DNA (the "stuffer" fragment) was removed. This creates room in the genome to clone a piece of foreign DNA (an insert) into the virus, which will then be replicated along with the viral genome. This means that we can use lambda GT10 to amplify a cloned piece of DNA and make many copies of it. Second, lambda GT10 has been modified to only use the lytic cycle when it is grown in an appropriate host. For this reason, when you grow the phage on a lawn of the appropriate strain of E. coli, the phage will lyse the bacteria and you will see a clear spot on the cloudy film of E. coli. A typical petri dish would look like this:

Figure 1
Plating out lambda GT10

This experiment is performed by adding phage to E. coli and then plating the E. coli by a pour plating technique (using soft LB media agar). Since the E. coli grow faster than the lambda GT10 can lyse them, a lawn of bacteria will grow first, creating a turbid film on top of the agar. Then the lambda GT10 begin to lyse E. coli and the turbidity is cleared. The result is clear spots (plaques) on a turbid background of E. coli. Each of these plaques represents a single lambda GT10 phage that infected a single E. coli when the lambda GT10 was originally added to the E. coli. By counting the number of plaques we can calculate how many phage were added to the E. coli before plating them out.

Phage are usually stored at a very high concentration. An accurate determination of the number of phage/ml in the stock (the titre of the stock) is important to its further use. In order to determine the titre, we need to dilute this phage stock. If there are too many phage on a plate, the plaques will overlap and you won't be able to count them accurately. To get an accurate determination of the titre we need to do serial dilutions of the stock. These are a series of one hundred fold dilutions:

Figure 2

In this figure, 10 ul of the stock solution is diluted 100 fold by putting it into 0.990 ml of SM buffer. Since this is a 100 fold dilution, the new solution is labeled 10-2 (i.e., its concentration is 1/100th that of the original stock). 10 ul of this new solution is then diluted to 1.000 ml, and this is labeled 10-4. 10 ul of this solution is again diluted 100 fold, and the final solution is labeled 10-6.

Note that the second and third dilutions are made from the first dilution. This means that any mistakes made in the first dilution will be magnified in subsequent dilutions. For this reason, careful pipeting is crucial to obtaining accurate results.

After diluting the stock in SM buffer, a sample of the one of the dilutions is incubated with E. coli. Soft agar at 45oC is then added and the mixture is plated out on LB agar plates. The next day you can calculate the number of phage in the original stock by counting the number of plaques on the plate and multiplying this number by the dilution used to make the plate.

An Example:

The following is an example of how this experiment will look:

You are give a sample of phage in SM media and you assume that the concentration of phage is somewhere between 106 and 108 phage per ml. You do serial dilutions of the phage stock as in Figure 2. Then you add 10 ul of the 10-4 dilution to 0.990 ml of E. coli and plate this out (total dilution of this sample is 10-4 X the final 100 fold dilution = 10-6). Then add 100 ul of the 10-6 dilution in 0.900 ml E coli and plate this out (total dilution is 10-6 X the final 10 fold dilution = 10-7). Finally, add 10 ul of the 10-6 dilution to 0.990 ml E. coli and plate this out (total dilution is 10-6 X the final 100 fold dilution = 10-8). The next day you check the plates for growth of plaques. The 10-6 plate has too many plaques to count. The 10-7 plate has 21 plaques. The 10-8 plate has 3 plaques. From this you conclude that the original phage stock had about 21 X 107 phage/ml, or 2 X 108 phage/ml.

Procedure:

Day one: During the lab period

1. You will be given a 20 ul sample of phage of unknown titre. Put your initials on the tube. Take three 1.5 ml eppendorf tubes and carefully dispense 0.990 ml of SM media in each under sterile conditions (i.e., with a bunson burner). Label the tubes 10-2, 10-4 and 10-6 and put your initials on each tube. Do serial dilutions of the phage as in Figure 2. Pipet very carefully and accurately. Small errors in pipetting will be magnified through each step of the dilution. Mix the solutions well between pipeting. *Keep all phage solutions on ice!* l Lambda GT10 will die if frozen or kept at room temperature for extended periods. Keep them away from your flame.

2. Dispense the E. coli solution that you have been given into three tubes. (Before you come to lab, an overnight culture of E. coli will have been spun down in a centrifuge and resuspended in 40% of its original volume with SM medium.) Put 0.990 ml E. coli in one culture tube and 0.900 ml E. coli in two culture tubes.

3.

4. Incubate the E. coli/phage mixtures at 37oC for 20 min. to allow the phage to adsorb to the E. coli.

5. While these are incubating, dispense 3.0 ml of 45oC LB top agar into each of three tubes and place them at 45oC. Label three LB agar plates 10-5, 10-6, 10-7.

6. Pour three ml of top agar into the 10-5 tube, mix quickly by inverting with parafilm, and rapidly pour it onto the 10-5 plate. Repeat for the 10-6 and 10-7 plates.

7. Allow the plates to sit flat on the bench until they solidify and then place them upside down at 37oC overnight. Store the tubes of phage (original stock and 10-2 dilution) in the refrigerator.

Day 2:
The next day before 4:00 P.M.

Count the number of plaques on each plate and calculate the titre of the original stock solution. Put these results into your lab report. Be sure to come in and check your results. The plates will be discarded by day 3.













If you have questions please send me a message at my Email address