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Team:BYU Provo/Notebook/SmallPhage/Springexp/Period2/Exp/5.20 Mutagen Concentration Experiment
From 2013.igem.org
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
- ddH2O; Thermo Taq buffer; dNTPs; primer; BI257; BI258; Taq polymerase
IV) Procedure
1) Isolating DNA
- 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.
- Boil for 12 minutes in the PCR machine.
- Remove the tubes from the PCR machine and shake the tube. Centrifuge it for 1 minutes at top speed.
- Keep on ice. DNA should be in the supernatant.
2) PCR
- To a centrifuge tube, add
- 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
- Label 3 PCR tubes "C" (Control), "5" (5 ul of phage stock), and "10" (10 ul of phage stock).
- Add 2 ul of template DNA from the supernatant of step 1 into their respective tubes (nothing in the control tube).
- Add 1.5 ul of TAQ Polymerase into the centrifuge tube (master mix).
- Pipette 48 ul of master mix into each PCR tube.
- 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.
