Team:TU Delft/protocols/colony PCR


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colony PCR


- 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


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
Primer 1 3 μL
Primer 2 3 μL
H20 19 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