Small Phage

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Revision as of 23:14, 20 May 2013

March

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experiment

April

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May

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5/20/13

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

T7 Minor Capsid Protein PCR

PCR Protocol for T7 Capsid Protein

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 PCR tube, add: o 40 ul ddH20 o 5 ul 10X TAQ buffer o 1.5 ul 10mMdNTP’s o 1 ul of each primer o 1 uL of template DNA (from supernatant of the step 1) • Mix well • Add 0.5 ul TAQ Polymerase • Keep on ice until placed in the PCR machine • Create a duplicate as a control, but do not add the template • 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 • If left overnight, keep at 4 C 3) Check with Agarose Electrophoresis • 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. • 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. • 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.

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June

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July

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@@ Line 51: / Line 51: @@
 ===5/20/13===
+LP, XL
+- We performed T7 Mutagen Concentration Test
+- We performed T7 Minor Capsid Protein PCR
+----
+'''T7 Minor Capsid Protein PCR'''
+PCR Protocol for T7 Capsid Protein
+)  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.
+) PCR
+•	To a PCR tube, add:
+o	40 ul ddH20
+o	5 ul 10X TAQ buffer
+o	1.5 ul 10mMdNTP’s
+o	1 ul of each primer
+o	1 uL of template DNA (from supernatant of the step 1)
+•	Mix well
+•	Add 0.5 ul TAQ Polymerase
+•	Keep on ice until placed in the PCR machine
+•	Create a duplicate as a control, but do not add the template
+•	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
+•	If left overnight, keep at 4 C
+)  Check with Agarose Electrophoresis
+•	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.
+•	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.
+•	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.
 ===5/22/13===