COLONY PCR:

Prepare mastermix according to your sample amount ( except Taq and template)

(For 1X sample preparation)

• dNTP 1 µL
• Buffer 5 µL
• MgCl2 3 µL
• **Forward Primer 0,5 µL
• **Reverse Primer 0,5 µL
• Template -
• Taq Polymerase 0,2 µL
• dH2O 39,8 µL
• TOTAL 50 µL

* You should add ingredients from largest amount to smallest amount.

* Before addition of primers and template you can do vortex.

** First you should pour ddH20 onto dried primers according to the amount written in the primer sheet then you should dilute (1:10 ) it in to new eppendorf(10 ml primer+ 90 ml ddH20)

Pour your samples into PCR tubes and add template that you pick from the petri with toothpick or tip and finally add taq polymerase. Then place your samples into the PCR machine and do regular PCR.

PCR Purification:

We used the protocol provided with the Qiagen kit .

• 3 volumes of Buffer QG is added to 1 volume of the PCR sample and mixed by vortexing.
• 1 volume of the INITIAL sample volume of isopropanol is added to the sample and mix.
• Place a QIAquick spin column in a provided 2 ml collection tube. Apply the sample to the QIAquick column with the pipette and centrifuge for 30–60 s.Discard flow-through. Place the QIAquick column back into the same tube
• Add 0.75 ml Buffer PE to the QIAquick column with the pipette and centrifuge for 30–60 s. Discard flow-through and place the QIAquick column back in the same tube.
• Centrifuge the column for an additional 1 min.
• Label the top of clean 1.7 ml microcentrifuge tube(s) with the name of your sample(s). Transfer QIAquick column(s) to the tube(s).
• To elute DNA, add 50 µl Buffer EB (10 mM Tris•Cl, pH 8.5) to the center of the QIAquick membrane. Let it stand for 1 min and centrifuge the column for 1 min.

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