Protocol/9

From 2010.igem.org

(Difference between revisions)
(New page: {{Team:Alberta/Head}} {{Team:Alberta/navbar|project=selected}} {{Team:Alberta/beginLeftSideBar}} {{Team:Alberta/endLeftSideBar}} {{Team:Alberta/beginRightSideBar}} {{Team:Alberta/endRig...)
Line 14: Line 14:
-
What you will need:
+
Reagents:
* Eppendorf tubes
* Eppendorf tubes
* Sterile toothpicks
* Sterile toothpicks
-
*PCR tubes (small tubes)
+
* PCR tubes (small tubes)
* 10X PCR buffer
* 10X PCR buffer
-
* 25 mM MgCl2­
+
* 25mM MgCl2­
* 5mM dNTPs
* 5mM dNTPs
* Taq polymerase
* Taq polymerase
-
* milliQ water
+
* MilliQ water
* PCR primers (specific for each reaction)  
* PCR primers (specific for each reaction)  
Line 28: Line 28:
Procedure:
Procedure:
-
 
+
* Set up one Eppendorf tube for each colony you are testing. Add 75uL MilliQ water to each tube.
-
* Set up one Eppendorf tube for each colony you are testing. Add 75 uL milliQ water to each tube.
+
* Label each colony you are testing by circling it and numbering it on the back of the agar plate.
* Label each colony you are testing by circling it and numbering it on the back of the agar plate.
-
* Remove 20-50% of the each colony with a sterile toothpick and twist the toothpick in the water in the corresponding tube. These tubes are your template tube. Use the same toothpick to streak for single colonies on an appropriate plate. Divide the plate in quarters – streak one colony per quarter
+
* Remove 20-50% of the each colony with a sterile toothpick and twist the toothpick in the water in the corresponding tube. These tubes are your template tube. Use the same toothpick to streak for single colonies on an appropriate plate. Divide the plate in quarters – streak one colony per quarter.
-
* Pipette 4 uL of each template into a corresponding PCR tube.
+
* Pipette 4uL of each template into a corresponding PCR tube.
-
* Make a master PCR mix using the following recipe: for each reaction, add
+
* Make a master PCR mix using the following recipe: for each reaction, add:
{|
{|
-
| 2.5 uL 10X PCR buffer
+
| 2.5uL 10X PCR buffer
|-
|-
-
| 2.5 uL 25 mM MgCl2
+
| 2.5uL 25mM MgCl2
|-
|-
-
| 1.0 uL dNTPs
+
| 1.0uL dNTPs
|-
|-
-
| 1.0 uL forward primer
+
| 1.0uL forward primer
|-
|-
-
| 1.0 uL reverse primer
+
| 1.0uL reverse primer
|-
|-
-
| 12.5 uL milliQ water
+
| 12.5uL milliQ water
|-
|-
-
| 0.5 uL Taq polymerase (remember to add enzyme last!)
+
| 0.5uL Taq polymerase  
|}
|}
-
 
+
*Remember to add enzyme last!
-
This recipe is for one reaction. If you are doing 10 reactions, multiply each volume by 11; you will need a little extra for pipetting error. Remember to mix your master mix thoroughly.
+
*This recipe is for one reaction. If you are doing 10 reactions, multiply each volume by 11; you will need a little extra for pipetting error. Remember to mix your master mix thoroughly.
-
 
+
* Add 21 uL of your master mix to each of the tubes containing template.
* Add 21 uL of your master mix to each of the tubes containing template.
* Load samples into PCR machine. Run program ‘Colony PCR’ or Col56e2 on the PTC 200 thermal cycler.
* Load samples into PCR machine. Run program ‘Colony PCR’ or Col56e2 on the PTC 200 thermal cycler.
 +
[[Team:Alberta/Notebook/protocols| Back]]
[[Team:Alberta/Notebook/protocols| Back]]
{{Team:Alberta/endMainContent}}
{{Team:Alberta/endMainContent}}

Revision as of 01:25, 26 October 2010

TEAM ALBERTA

Protocol 9: Colony PCR


Reagents:

  • Eppendorf tubes
  • Sterile toothpicks
  • PCR tubes (small tubes)
  • 10X PCR buffer
  • 25mM MgCl2­
  • 5mM dNTPs
  • Taq polymerase
  • MilliQ water
  • PCR primers (specific for each reaction)


Procedure:

  • Set up one Eppendorf tube for each colony you are testing. Add 75uL MilliQ water to each tube.
  • Label each colony you are testing by circling it and numbering it on the back of the agar plate.
  • Remove 20-50% of the each colony with a sterile toothpick and twist the toothpick in the water in the corresponding tube. These tubes are your template tube. Use the same toothpick to streak for single colonies on an appropriate plate. Divide the plate in quarters – streak one colony per quarter.
  • Pipette 4uL of each template into a corresponding PCR tube.
  • Make a master PCR mix using the following recipe: for each reaction, add:
2.5uL 10X PCR buffer
2.5uL 25mM MgCl2
1.0uL dNTPs
1.0uL forward primer
1.0uL reverse primer
12.5uL milliQ water
0.5uL Taq polymerase
  • Remember to add enzyme last!
  • This recipe is for one reaction. If you are doing 10 reactions, multiply each volume by 11; you will need a little extra for pipetting error. Remember to mix your master mix thoroughly.
  • Add 21 uL of your master mix to each of the tubes containing template.
  • Load samples into PCR machine. Run program ‘Colony PCR’ or Col56e2 on the PTC 200 thermal cycler.


Back

Retrieved from "http://2010.igem.org/Protocol/9"