Team:Paris Saclay/protocols/pcr

From 2013.igem.org

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(→PCR for the genomic DNA of bacteria)
 
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1. In a cold PCR tube (eppendrof), pipette in order the required substances in the Table 1.
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1. In a cold PCR tube (Eppendorf), pipet in the presented order the solutions listed in Table 1.
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to do
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{| class="wikitable centre" width="80%"
 +
|+
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|-
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! scope=col | Component
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! scope=col | Volume/50µL reaction
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! scope=col | Volume/20µL reaction
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|-
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| width="33%" |
 +
H<sub>2</sub>O
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| width="34%" |
 +
Add to 50 μl
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| width="33%" |
 +
Add to 20 μl
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|-
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| width="33%" |
 +
5x Phusion HF Buffer High-fidelity
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| width="34%" |
 +
10 μl
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| width="33%" |
 +
4 μl
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|-
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| width="33%" |
 +
dNTPs 10 mM
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| width="34%" |
 +
1 μl
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| width="33%" |
 +
0.4 μl
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|-
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| width="33%" |
 +
Primer A
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| width="34%" |
 +
x μl
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| width="33%" |
 +
x μl
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|-
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| width="33%" |
 +
Primer B
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| width="34%" |
 +
x μl
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| width="33%" |
 +
x μl
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|-
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| width="33%" |
 +
Template DNA: Genomic DNA
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| width="34%" |
 +
x μl
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| width="33%" |
 +
x μl
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|-
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| width="33%" |
 +
Phusion DNA polymerasse
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| width="34%" |
 +
0.5 μl
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| width="33%" |
 +
0.2 μl
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 +
 
 +
|}
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to do
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{| class="wikitable centre" width="80%"
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|+
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|-
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! scope=col | Cycle step
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! scope=col | Temperature
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! scope=col | Time
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! scope=col | Cycle
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|-
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| width="33%" |
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Initial denaturation
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| width="34%" |
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95 - 98 °C
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| width="33%" |
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30 s – 3 min
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| width="34%" |
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1
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|-
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| width="33%" |
 +
Denaturation
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| width="34%" |
 +
95 - 98 °C
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| width="33%" |
 +
10 – 30 s
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| width="34%" |
 +
25-30
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|-
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| width="33%" |
 +
Annealing
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| width="34%" |
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variable
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| width="33%" |
 +
30 s
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| width="34%" |
 +
25-30
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|-
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| width="33%" |
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Extension
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| width="34%" |
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72°C
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| width="33%" |
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30 s – 2 min
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| width="34%" |
 +
25-30
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|-
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| width="33%" |
 +
Final extension
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| width="34%" |
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72°C
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| width="33%" |
 +
10min
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| width="34%" |
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1
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|-
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| width="33%" |
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Final extension
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| width="34%" |
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4-8°C
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| width="33%" |
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hold
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| width="34%" |
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1
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|}
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3. Determine the number of oligo by electrophoresis.
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3. Verify correct amplification by agarose gel electrophoresis.
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to do
 
{{Team:Paris_Saclay/incl_fin}}
{{Team:Paris_Saclay/incl_fin}}

Latest revision as of 09:24, 4 October 2013

Navigation Home Team Project Labwork Notebook Human practices

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.


Component Volume/50µL reaction Volume/20µL reaction

H2O

Add to 50 μl

Add to 20 μl

5x Phusion HF Buffer High-fidelity

10 μl

4 μl

dNTPs 10 mM

1 μl

0.4 μl

Primer A

x μl

x μl

Primer B

x μl

x μl

Template DNA: Genomic DNA

x μl

x μl

Phusion DNA polymerasse

0.5 μl

0.2 μl



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

10min

1

Final extension

4-8°C

hold

1

3. Verify correct amplification by agarose gel electrophoresis.



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