Protocol/9

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Protocol 9: Colony PCR


What you will need:
* Eppendorf tubes
* Sterile toothpicks
*PCR tubes (small tubes)
* 10X PCR buffer
* 25 mM 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 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.
* 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.
* Make a master PCR mix using the following recipe: for each reaction, add
{|
| 2.5 uL 10X PCR buffer
|-
| 2.5 uL 25 mM MgCl2
|-
| 1.0 uL dNTPs
|-
| 1.0 uL forward primer
|-
| 1.0 uL reverse primer
|-
| 12.5 uL milliQ water
|-
| 0.5 uL 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.

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