Team:TU Delft/protocols/colony PCR

From 2010.igem.org

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==colony PCR==
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=colony PCR=
''Materials:''
''Materials:''
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- 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.
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- 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.
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- Primer solutions 5 mol/mL
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- primer solutions 5 mol/mL
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- PCR machine
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|Taq PCR Master Mix (Qiagen)
|Taq PCR Master Mix (Qiagen)
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|25 μL
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|12.5 μL
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|-
|Primer 1
|Primer 1
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|3 μL
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|1.5 μL
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|-
|Primer 2
|Primer 2
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|3 μL
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|1.5 μL
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|-
|H20
|H20
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|19 μL
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|9.5 μL
|}
|}

Revision as of 11:11, 12 September 2010

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