Team:BYU Provo/Notebook/SmallPhage/Summerexp/9.13 Mutagen Concentration Test

Small Phage

March-April

May-June

July-August

September-October

To mutate T7 phage for different capsid sizes.

II) Expected Outcome

• A decrease in phage viability with increasing mutagen concentration.

• A few random mutations that produce smaller and larger phage that can be isolated.

III) Reagents Used

• 5-bromodeoxyuridine stock in freezer labeled T7 (10mg/mL)

• Uracil solution (2.5mg/mL)

• Adenine solution (5mg/mL)

• 8.24 phage stock

• x2 top agar

• LB

IV) Procedure

1) Overnight (The day before) (9.13)

• Add 5mL of LB into a test tube and add 1 colony of E. Coli B into the tube using a wooden stick.

2) Applying the mutagen (9.14)

• Label 4 test tubes C, 0, 500, 1000. Add 9.8mL of LB and 40ul of adenine solution into each test tube. Then add 200ul of E coli B overnight into each test tube. Incubate on the shaker at 37 Celsius.

• Remove all the test tubes off the shaker after 2 hours. Add 40ul of adenine and 80ul of uracil to each test tube. Also add the corresponding amount in ul of 5-bromodeoxyuridine, a mutagen, to each test tube. (Ex: Add 500ul of mutagen to tube labeled 500). Don't add mutagen to C.

• Place all the tubes on the shaker at 37 Celsius.

• After 20 minutes, take only the tube labeled C from the shaker. Take 1mL from this tube and pipette it into a cuvette labeled C. Using 1mL of LB in a cuvette, blank the spectrophotometer at 600 OD. Then measure the absorbance of the cuvette labeled C. (In the actual procedure, 2 readings were done on the tube labeled C to check for consistency.

• Remove the other three tubes after 30 minutes of incubation and add 900uL of T7 phage from the 8.24 stock to each tube, except C. From this point on, tube C is irrelevant and can be discarded. (There should be a 1:10 phage to bacteria concentration. The calculations are as follows: The spectrophotometer reading was 0.315, which indicates there are 1.58E8 bacteria/ml. Because there are 10ml in each tube, there is roughly 1.585E9 bacteria per tube. This means that we need 1.58E8 phage added to each tube. Because the 8.24 stock is concentrated at 1.5E8 phage/ml [3E6 phage/20ul], 1053uL of phage stock will provide 1.58E8 phage. Only 900uL of phage stock was added to account for estimation error.)

• Incubate all the tubes on the shaker at 37 Celsius for 80 minutes.

• Remove all the tubes and add 1mL of chloroform to each. Gently shake each tube and centrifuge it at 4000rpm for 10 minutes at 7 Celsius. Remove the supernatant from each tube with a pipette and place it in a new tube with the same label. Be careful not to get the chloroform or bacteria when you remove the supernatant.

• Store the supernatants at 4 Celsius.

3) Cesium Chloride Gradients

• The phage purification team ran 3 cesium chloride gradients:

- Wild type

- One that selects for small phage

- One that selects for large phage

• For their procedure, go here (ADD LINK)

4) Spot Test of Gradient Samples

• The small phage and large phage gradients were divided into 15 2ml eppendorf tubes each.

• We created a -1, -2, and -4 dilution series for each tube.

• We then spotted 5mL from each tube onto plates that had 1x top agar.

V) Results

2) Applying the mutagen

• The OD reading was 0.435A, which indicates there are 2.175E8 bacteria/ml. Because there are 10ml in each tube, there is roughly 2.175E9 bacteria per tube.

VI) Conclusion