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From 2010.igem.org

colony PCR

Materials:

- Taq PCR Master Mix (Qiagen) is premixed solution containing Taq DNA Polymerase, PCR Buffer and dNTPs. The solution provides a final concentration of 1.5 mM MgCl2 and 200 μM each dNTP.

- primer solutions 5 mol/mL

- PCR machine

Protocol:

First make sure that there is a PCR machine available for you. Take the solutions from the freezer and thaw on ice. Preparation of reaction mixture:

1. Gently vortex and briefly centrifuge all solutions after thawing

2. Keep solutions on ice

3. Make a pre-mix for the amount of colonies (to be analyzed) + 1, add to an Eppendorf tube:

1× pre-mix

Component	Sample
Taq PCR Master Mix (Qiagen)	12.5 μL
Primer 1	1.5 μL
Primer 2	1.5 μL
H20	9.5 μL

4. Add 50 μL of pre-mix to each PCR tube.

5. Prick a sterile toothpick into a colony, dip it into a PCR tube, put it into a 15 mL culture tube containing 5 mL LB medium + antibiotics and then grow overnight for mini-prep cultures and -80 °C stocks. Repeat this for all the colonies. Incubate the mini-prep cultures at 37 °C.

6. Keep everything ice cold until you put the tubes in the preheated PCR machine

PCR program:

Step	Annealing Temperature	Time, min:sec	Number of cycles
Initial denaturation	94 °C	2:00	1
Denaturation	94 °C	1:00	30
Annealing	x °C *	0:45	30
Extension	72 °C	1:00	30
Final Extension	72 °C	5:00	1

* Calculate the optimal annealing temperature = 3x G/C + 2x A/T