Colony PCR

From 2011.igem.org

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(Created page with "[https://2011.igem.org/Team:Panama/Protocols/Wetlab '''Back''']")
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'''Materials:'''
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Agar plate with colonies
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PCR mastermix
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Forward primers
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Reverse primers
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Molecular grade H2O
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agar plate with appropriate antibiotic
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'''Procedure:'''
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1. Prepare a stock solution using per colony
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12.5ul mastermix
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2.5ul forward primer
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2.5ul reverse primer
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7.5ul Molecular grade H2O
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2. aliquot in 0.6 ml PCR tubes 25ul of the stock solution. Do this for every colony you intend to pick.
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3. using a micropipette with a yellow tip tap the colony you want to pick and dip the tip in one of the PCR tubes.
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4. streak the colony onto an agar plate to save the colony.
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5. run your PCR program.
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'''we used:'''
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1. 94°C - 5.00 min
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2. 94°C - 30 sec
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51°C -  1.00 min
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72°C - 3.00 min
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repeat 3-5 for 40 cycles
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72°C - 10.00 min
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4°C - forever
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[https://2011.igem.org/Team:Panama/Protocols/Wetlab '''Back''']
[https://2011.igem.org/Team:Panama/Protocols/Wetlab '''Back''']

Revision as of 02:39, 29 September 2011

Materials:

Agar plate with colonies

PCR mastermix

Forward primers

Reverse primers

Molecular grade H2O

agar plate with appropriate antibiotic

Procedure:

1. Prepare a stock solution using per colony

12.5ul mastermix

2.5ul forward primer

2.5ul reverse primer

7.5ul Molecular grade H2O

2. aliquot in 0.6 ml PCR tubes 25ul of the stock solution. Do this for every colony you intend to pick.

3. using a micropipette with a yellow tip tap the colony you want to pick and dip the tip in one of the PCR tubes.

4. streak the colony onto an agar plate to save the colony.

5. run your PCR program.

we used:

1. 94°C - 5.00 min

2. 94°C - 30 sec

51°C - 1.00 min

72°C - 3.00 min

repeat 3-5 for 40 cycles

72°C - 10.00 min

4°C - forever

Back

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