Team:TU Delft/protocols/PCR

From 2010.igem.org

(Difference between revisions)
(New page: ==PCR== ''Materials:'' - Taq PCR Master Mix (Qiagen) is premixed solution containing Taq DNA Polymerase, PCR Buffer and dNTPs. The solution provides a final concentration of 1.5 mM MgCl...)
 
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==PCR==
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=PCR=
''Materials:''
''Materials:''
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- Taq PCR Master Mix (Qiagen) is premixed solution containing Taq DNA Polymerase, PCR Buffer and dNTPs. The solution provides a final concentration of 1.5 mM MgCl2 and 200 μM each dNTP.
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- Pfx Polymerase (Invitrogen)  
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- Primer solutions 5 mol/mL
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- 10x Pfx Bufer (Invitrogen)
 +
 
 +
-      Enhancer (Invitrogen)
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 +
- 50 mM MgSO4
 +
 
 +
- 10 mM dNTPs
 +
 
 +
- Primer solutions 5 pmol/μL
- Template DNA (plasmid at 50 pg – 1 ng/μL)
- Template DNA (plasmid at 50 pg – 1 ng/μL)
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|Sample
|Sample
|-
|-
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|Taq PCR Master Mix (Qiagen)
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|Pfx polymerse
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|25 μL
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|0.6 μL
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|-
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|10x Pfx buffer
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|5 μL
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|-
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|Enhancer
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|5 μL
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|-
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|10 mM dNTPs
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|1.5 μL
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|-
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|50 mM MgSO4
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|1 μL
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|Primer 1
|Primer 1
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|DNA template
|DNA template
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|5 μL
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|1 μL
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|H20
|H20
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|59 μL
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|29.9 μL
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5. Place samples from the ice in a thermal cycler preheated to 95 °C and start the previously programmed PCR.
5. Place samples from the ice in a thermal cycler preheated to 95 °C and start the previously programmed PCR.
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PCR program:
PCR program:
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|Initial denaturation
|Initial denaturation
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|94 °C  
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|x °C *
|2:00
|2:00
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|1
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|-
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|Annealing
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|95 °C
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|2:00
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|1
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|-
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|Extension
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|68 °C
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|1:00
|1
|1
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|Denaturation
|Denaturation
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|94 °C  
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|95 °C  
|1:00
|1:00
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|30
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|25
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|Annealing
|Annealing
|x °C *
|x °C *
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|0:45
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|1:00
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|30
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|25
|-
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|Extension
|Extension
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|72 °C
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|68 °C
|1:00
|1:00
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|30
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|25
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|Final Extension
|Final Extension
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|72 °C
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|68 °C
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|5:00
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|10:00
|1
|1
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* Calculate the optimal annealing temperature = 3x G/C + 2x A/T
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[[*]] Calculate the optimal annealing temperature = 3x G/C + 2x A/T

Latest revision as of 11:09, 12 September 2010

PCR

Materials:

- Pfx Polymerase (Invitrogen)

- 10x Pfx Bufer (Invitrogen)

- Enhancer (Invitrogen)

- 50 mM MgSO4

- 10 mM dNTPs

- Primer solutions 5 pmol/μL

- Template DNA (plasmid at 50 pg – 1 ng/μL)


Protocol:

First make sure that there is a PCR machine available for you. Take the solutions from the freezer and thaw them on ice. Preparation of reaction mixture:

1. Gently vortex and briefly centrifuge all solutions after thawing

2. Keep solutions on ice

3. Add to a thin walled PCR tube, on ice, for a 100 μL reaction:

1× pre-mix

Component Sample
Pfx polymerse 0.6 μL
10x Pfx buffer 5 μL
Enhancer 5 μL
10 mM dNTPs 1.5 μL
50 mM MgSO4 1 μL
Primer 1 3 μL
Primer 2 3 μL
DNA template 1 μL
H20 29.9 μL


4. Gently vortex the sample and briefly centrifuge (5 sec) to collect all droplets at the bottom of the tube

5. Place samples from the ice in a thermal cycler preheated to 95 °C and start the previously programmed PCR.

PCR program:

Step Annealing Temperature Time, min:sec Number of cycles
Initial denaturation x °C * 2:00 1
Annealing 95 °C 2:00 1
Extension 68 °C 1:00 1
Denaturation 95 °C 1:00 25
Annealing x °C * 1:00 25
Extension 68 °C 1:00 25
Final Extension 68 °C 10:00 1


* Calculate the optimal annealing temperature = 3x G/C + 2x A/T

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