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• iGEM 2013

5.20 T7 Minor Capsid Protein PCR

I) Purpose

To isolate the T7 minor capsid protein

II) Expected Outcome

We expect that we will have isolated the T7 minor capsid protein. This can be visualized by gel electrophoresis and there should be one band that matches the number of base pairs in the minor capsid protein.

III) Reagents Used

ADD

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 10mMdNTP'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.