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Team:TU Delft/protocols/colony PCR
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Revision as of 06:24, 2 August 2010
colony PCR
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 |
| 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
