Team:BYU Provo/Notebook/SmallPhage/Springexp/Period2/Exp/5.20 Mutagen Concentration Experiment

From 2013.igem.org

(Difference between revisions)

Revision as of 18:02, 21 May 2013

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

ADD

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

@@ Line 19: / Line 19: @@
 '''IV) Procedure'''
-- From Put 5 ul of phage stock and 45 ul of ddH20 in one PCR tube and 10 ul of phage stock and 40 ul of ddH20 in another PCR tube.
+) Applying the mutagen (5.20)
-- Boil for 12 minutes in the PCR machine.
+: - 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.
-- Remove the tubes from the PCR machine and shake the tube.  Centrifuge it for 1 minutes at top speed.
+: - 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
-- Keep on ice.  DNA should be in the supernatant.
+: - 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.
-) PCR
+: ''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.''
-- To a centrifuge tube, add
+: - Let the bacterial suspension incubate with mutagen for approximately 15min.
-: 120 ul ddH20
-: 15 ul 10X TAQ Buffer
-: 4.5 ul 10mM dNTP's
-: 3 ul of forward primer
-: 3 ul of reverse primer
-- Mix well
+: - 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.
-- Label 3 PCR tubes "C" (Control), "5" (5 ul of phage stock), and "10" (10 ul of phage stock).
+: - Incubate phage on shaker at 37C for approximately 20min.
-- Add 2 ul of template DNA from the supernatant of step 1 into their respective tubes (nothing in the control tube).
+: - 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.
-- Add 1.5 ul of TAQ Polymerase into the centrifuge tube (master mix).
+: - The stock solutions are now stored in 4C
-- Pipette 48 ul of master mix into each PCR tube.
+) Spot test
-- Run 35 cycles with temperatures of 95 C, 50 C, and 72 C with an extension time of 1.5 minutes.
-- Leave overnight at 4 C.
-- Remove and place in the freezer.
 {{TeamBYUProvoFooter}}