Small Phage

From 2013.igem.org

(Difference between revisions)
(→5/20/13)
(→5/20/13)
Line 60: Line 60:
'''T7 Minor Capsid Protein PCR'''
'''T7 Minor Capsid Protein PCR'''
-
PCR Protocol for T7 Capsid Protein
+
'''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) Results'''
1)  Isolating DNA
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.
- 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.
Line 72: Line 85:
2) PCR
2) PCR
-
• To a PCR tube, add:
+
 
-
o 40 ul ddH20
+
- To a centrifuge tube, add
-
o 5 ul 10X TAQ buffer
+
: 120 ul ddH20
-
o 1.5 ul 10mMdNTP’s
+
: 15 ul 10X TAQ Buffer
-
o 1 ul of each primer
+
: 4.5 ul 10mMdNTP's
-
o 1 uL of template DNA (from supernatant of the step 1)
+
: 3 ul of forward primer
-
• Mix well
+
: 3 ul of reverse primer
-
• Add 0.5 ul TAQ Polymerase
+
- Mix well
-
• Keep on ice until placed in the PCR machine
+
 
-
• Create a duplicate as a control, but do not add the template
+
- Label 3 PCR tubes "C" (Control), "5" (5 ul of phage stock), and "10" (10 ul of phage stock).
-
• Prewarm the PCR machine to 94 C
+
 
-
• Run 35-40 cycles with temperatures of 94 C, 50 C, and 72 C and an extension time of 1.5 minutes
+
- Add 2 ul of template DNA from the supernatant of step 1 into their respective tubes (nothing in the control tube).
-
• If left overnight, keep at 4 C
+
 
-
3)  Check with Agarose Electrophoresis
+
- Add 1.5 ul of TAQ Polymerase into the centrifuge tube (master mix).
-
• To make a standard 1% gel use 75 ml of 1X TAE and 0.75 grams of agarose (regular agarose, not low melt).  Put it into the microwave for about 90 seconds or until the agarose is completely dissolved.  Pulsing the microwave may be necessary to prevent boiling over
+
 
-
• Add 12 ul of 1mg/ml ethidium bromide and swirl to mix.  Be sure to wear gloves.
+
- Pipette 48 ul of master mix into each PCR tube.
-
• Allow flask to cool so that the glass feels warm, not burning hot.  Pour the liquid onto the gel bed and let it cool.  Insert the appropriate sample comb (need 3 slots).
+
 
-
• Add loading dye to each PCR product.  Do this by adding 6 ul of 10X loading dye to a 50ul PCR.
+
- Run 35 cycles with temperatures of 95 C, 50 C, and 72 C with an extension time of 1.5 minutes.
-
• Move the gel into the proper orientation (DNA runs towards the red electrode) in the gel box, cover the gel with 1X TAE buffer and load 4-5 ul of each of the PCR product.  Add a DNA ladder as a reference.  Turn on the power supply and run at 150-175 volts.  It will take about 15-30 to complete.
+
 
-
• Visualize the gel on the Alphaimager.  Print off the results and log in your notebook.
+
- Leave overnight at 4 C.
 +
 
 +
- Remove and place in the freezer.
===5/22/13===
===5/22/13===

Revision as of 23:35, 20 May 2013

Contents

March

3/15/13

3/18/13

3/20/13

3/22/13

3/25/13

3/27/13

3/29/13

experiment

April

4/1/13

4/4/13

4/5/13

4/8/13

4/10/13

4/12/13

4/15/13

May

5/1/13

5/3/13

5/6/13

5/8/13

5/10/13

5/13/13

5/15/13

5/17/13

5/20/13

LP, XL - We performed T7 Mutagen Concentration Test - We performed T7 Minor Capsid Protein PCR


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) Results

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.

5/22/13

5/24/13

5/27/13

5/29/13

5/31/13

June

6/3/13

6/5/13

6/7/13

6/10/13

6/12/13

6/14/13

6/17/13

6/19/13

6/21/13

6/24/13

6/26/13

6/28/13

July

7/1/13

7/3/13

7/5/13

7/8/13

7/10/13

7/12/13

7/15/13

7/17/13

7/19/13

7/22/13

7/24/13

7/26/13

7/29/13

7/31/13

August

8/2/13

8/5/13

8/7/13

8/9/13

8/12/13

8/14/13

8/16/13

8/19/13

8/21/13

8/23/13

8/26/13

8/28/13

8/30/13

September

9/2/13

9/4/13

9/6/13

9/9/13

9/11/13

9/13/13

9/16/13

9/18/13

9/20/13

9/23/13

9/25/13

9/27/13

9/30/13

Retrieved from "http://2013.igem.org/Small_Phage"