|
- Overview
- Winter
- Spring
- Summer
- Fall
|
5.20 Mutagen Concentration Experiment
I) Purpose
- To optimize 5-bromodeoxyuridine concentration for T7 phage
II) Expected Outcome
- - Settle procedure for inducing random mutations in phage.
- - 5-bromodeoxyuridine concentration will affect phage viability, which will be seen in the different number of plaques formed. 5-bromodeoxyuridine should aslo generate random mutations that produce relatively smaller phage.
III) Reagents Used
- 5-bromodeoxyuridine stock in freezer labeled T7; various bottles of uncontaminated LB; BL21 overnight
IV) Procedure
1) Applying the mutagen (5.20)
- - From 5.13 Determining E coli concentration with spectrophotometer, we know that the concentration of E coli BL21 in a liquid overnight culture is about 3.12E8 cell/mL. We need to dilute it get our wanted E coli concentration.
- - Label 5 centrifuge tubes (15mL) 0, 10, 50, 100, and 200. To each of these test tubes, add 1.5mL of E coli overnight and 8.5mL of LB to make it a total of 10mL. This will give us approximately 5E7 cell/mL or 5E8 cell/test tube
- - To the five centrifuge tubes, add 0, 10, 50, 100, and 200 uL of 5-bromodeoxyuridine. The volume of the mutagen should correspond to the label on the test tube. Because 5-bromodeoxyuridine stock is a 10mg/mL aqueous solution, the final mutagen concentration in the five test tubes will be 0, 10, 50, 100, and 200 ug/mL.
- - Let the bacterial suspension incubate with mutagen for approximately 15min.
- - 6uL of 5.3 phage stock was added to each centrifuge tube. From 5.15 Titer Test on 5.3 T7 new Phage Stock, we know that 5.3 phage stock has 7E8 particle/20uL. Thus, to each of the test tubes, we are adding approximately 2.1E8 particle. This gives us a multiplicity of infection lower than 0.5 particles per cell.
- - Incubate phage on shaker at 37C for approximately 20min.
- - Liquid culture from the five test tubes is then centrifuged at 4000 rpm for 10 minutes to pellet cell debris. Supernatant containing phage particles is removed and 1mL of chloroform is added destroy any remaining E coli cells.
- - The stock solutions are now stored in 4C
2) Spot test (5.22)
- - Spot test was performed following procedures similar to those in 5.3 T7 Phage Amplification/Purification
3) Preliminary selection test (5.24)
- - Preliminary selection test was performed using x8 agar; -6, -7, and -8 dilutions; and procedure similar to those in 5.3 T7 Phage Selection Method Test
V) Results
1) Applying mutagen
- Phage did not amplify in the liquid culture with 5-bromodeoxyuridine. Calculations revealed that the initial concentration of phage in liquid culture should be approximately 2.5E7 phage particle/mL. This is on the same scale as the final phage concentration as revealed by the spot test below.
2) Spot test
- Lower concentration of mutagen seen to stimulate phage production, while higher concentration inhibits
3) Preliminary selection test
- Only one or two plaques are seen on each plate. The resolution of the following photo does not allow showing.
VI) Conclusion
ADD
|
"
