Team:Paris Saclay/Protocols/PCR for bacterial culture

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='''PCR for bacterial culture'''=
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{{Team:Paris_Saclay/protocols_header}}
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=PCR for bacterial culture=
:1. In a cold PCR tube (Eppendorf), pipet in the presented order the solutions listed in Table 1.
:1. In a cold PCR tube (Eppendorf), pipet in the presented order the solutions listed in Table 1.
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Add to final volume
Add to final volume
| width="25%" |
| width="25%" |
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37.75μl (50μl final)
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27.75μl (50μl final)
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|-
| Tp Green 5X
| Tp Green 5X
| 5X
| 5X
| 1X
| 1X
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| 5μl
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| 10μl
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|-
| dNTPs  
| dNTPs  
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| 200μM
| 200μM
| 1μl
| 1μl
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|-
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| MgCl<sub>2</sub>
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| -
 +
| -
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| 4μl
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|-
| Primer A
| Primer A
| 1mM
| 1mM
| 10μM
| 10μM
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| 2.5μl
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| 2μl
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|-
| Primer B
| Primer B
| 1mM
| 1mM
| 10μM
| 10μM
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| 2.5μl
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| 2μl
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|-
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| Template DNA: Genomic DNA
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| Culture
| -
| -
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| 1μl
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| -
-
| 1μl
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| 2μl
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|-
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| GoTaq DNA polymerasse
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| GoTaq DNA polymerase
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| -
| -
| -
| 0.25μl
| 0.25μl
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| 0.25μl
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|-
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| DMSO
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| -
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| -
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| 1.5μl
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<span style="color:red">Note: enzyme is added last, and dNTP second from last. 48μl of mixture is inserted in a PCR tube before adding 2μl of culture.</span>
:2. Program the PCR machine according to the Table 2.
:2. Program the PCR machine according to the Table 2.
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:3. Verify correct amplification by agarose gel electrophoresis.
:3. Verify correct amplification by agarose gel electrophoresis.
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[https://2014.igem.org/Team:Paris_Saclay/Protocols Back to the Protocols]
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{{Team:Paris_Saclay/protocols_footer}}

Latest revision as of 12:59, 5 August 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

Note: enzyme is added last, and dNTP second from last. 48μl of mixture is inserted in a PCR tube before adding 2μl of culture.

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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