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From 2010.igem.org

Today we colony PCR'd some of the transformants presumably containing the BioBricks we will be using for the rest of the Summer. We hope for the best and to have the results in over 1 hour!

The protocol followed for colony PCR is as follows:

Materials:

- Taq PCR Master Mix (Qiagen) is a 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

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)	25 μL
G00100	3 μL
G00101	3 μL
H20	29 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
Extension	72 °C	5:00	1

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

The ligation products of yesterday were transformed into our awesomely competent cells.