Team:Paris Saclay/Protocols/PCR for the genomic DNA of bacteria

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=PCR for the genomic DNA of bacteria=

:1. In a cold PCR tube (Eppendorf), pipet in the presented order the solutions listed in Table 1.

{| class="wikitable centre" width="80%"
|+ 
|-
! scope=col | Component
! scope=col | Initial concentration
! scope=col | Final concentration
! scope=col | Example for 50μl
|-
| width="25%" |
H<sub>2</sub>O
| width="25%" |
-
| width="25%" |
Add to final volume
| width="25%" |
37.75μl (50μl final)
|-
| 5x Phusion HF Buffer High-fidelity
| 10X
| 1X
| 5μl
|-
| dNTPs 
| 10mM
| 200μM
| 1μl
|-
| Primer A
| 100μM
| 0.5μM
| 2.5μl
|-
| Primer B
| 100μM
| 0.5μM
| 2.5μl
|-
| Template DNA: Genomic DNA 
| -
| 1μl
| 1μl
|-
| Phusion DNA polymerasse
| -
| 0.25μl
| 0.25μl

|}

:2. Program the PCR machine according to the Table 2.

{| class="wikitable centre" width="80%"
|+ 
|-
! scope=col | Cycle step
! scope=col | Temperature
! scope=col | Time
! scope=col | Cycle
|-
| width="25%" |
Initial denaturation
| width="25%" |
95 - 98°C
| width="25%" |
30 s - 3 min
| width="25%" |
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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