Team:Paris Saclay/Protocols/PCR for bacterial culture

From 2014.igem.org

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Revision as of 12:02, 29 July 2014

PCR for bacterial culture

1. In a cold PCR tube (Eppendorf), pipet in the presented order the solutions listed in Table 1.
Component Initial concentration Final concentration Example for 50μl

H2O

-

Add to final volume

27.75μl (50μl final)

Tp Green 5X 5X 1X 10μl
dNTPs 10mM 200μM 1μl
MgCl2 - - 4μl Primer A 1mM 10μM 2μl
Primer B 1mM 10μM 2μl
Culture - - 2μl
GoTaq DNA polymerase - - 0.25μl
DMSO - - 1.5μl
2. Program the PCR machine according to the Table 2.
Cycle step Temperature Time Cycle

Initial denaturation

95 - 98°C

30 s - 3 min

1

Denaturation 95 - 98°C 10 - 30 s 25 - 30
Annealing variable 30 s 25 - 30
Extension 72°C 30 s - 2 min 25-30
Final extension 72°C 10 min 1
Final extension 4 - 8°C hold 1
3. Verify correct amplification by agarose gel electrophoresis.

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