Team:Paris Saclay/protocols/pcr

From 2013.igem.org

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(Created page with "{{Team:Paris_Saclay/incl_debut_generique}} = '''PCR for the genomic DNA of bacteria''' = 1. In a cold PCR tube (eppendrof), pipette in order the required substances in the Tabl...")
(→PCR for the genomic DNA of bacteria)
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{| class="wikitable centre" width="80%"
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|+
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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%" |
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H2O
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| width="34%" |
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Add to 50 μl
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| width="33%" |
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Add to 20 μl
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|-
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| width="33%" |
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5x Phusion HF Buffer High-fidelity
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| width="34%" |
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10 μl
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| width="33%" |
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4 μl
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|-
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| width="33%" |
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10 mM dNTPs
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| width="34%" |
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1 μl
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| width="33%" |
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0.4 μl
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|-
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| width="33%" |
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Primer A
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| width="34%" |
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x μl
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| width="33%" |
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x μl
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|-
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| width="33%" |
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Primer B
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| width="34%" |
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x μl
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| width="33%" |
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x μl
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|-
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| width="33%" |
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Template DNA: Genomic DNA of bacteria E. coli DH5α
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| width="34%" |
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x μl
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| width="33%" |
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x μl
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|-
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| width="33%" |
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Phusion DNA polymerasse
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| width="34%" |
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0.5 μl
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| width="33%" |
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0.2 μl
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|}
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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%" |
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Denaturation
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| width="34%" |
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95 - 98 °C
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| width="33%" |
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10 – 30 s
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| width="34%" |
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25-30
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|-
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| width="33%" |
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Annealing
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| width="34%" |
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x °C
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| width="33%" |
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30 s - 1 min
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| width="34%" |
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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%" |
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25-30
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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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72°C
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| width="33%" |
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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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|}
3. Determine the number of oligo by electrophoresis.
3. Determine the number of oligo by electrophoresis.
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to do
 
{{Team:Paris_Saclay/incl_fin}}
{{Team:Paris_Saclay/incl_fin}}

Revision as of 20:25, 23 September 2013

Navigation Home Team Project Labwork Notebook Human practices

PCR for the genomic DNA of bacteria

1. In a cold PCR tube (eppendrof), pipette in order the required substances in the 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

10 mM dNTPs

1 μl

0.4 μl

Primer A

x μl

x μl

Primer B

x μl

x μl

Template DNA: Genomic DNA of bacteria E. coli DH5α

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

x °C

30 s - 1 min

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. Determine the number of oligo by electrophoresis.



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