From 2011.igem.org
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| - | == PCR protocol ==
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| - | PCR reaction components:
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| - | a. Enzyme
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| - | b. Forward primer
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| - | c. Reverse primer
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| - | d. dNTPs
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| - | e. template DNA
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| - | f. buffer
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| - | g. MilliQ or distilled water
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| - | Amounts per one reaction (100μl)
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| - | Taq+ pFU (μl) Phusion (μl)
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| - | enzyme 0,5 0,5
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| - | Forward primer 2,5 5
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| - | Reverse primer 2,5 5
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| - | dNTP 4 4
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| - | Template 1 1
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| - | Buffer 10 20
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| - | water 79,5 64,5
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| - | Master mix
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| - | a. Prepare master mix for the amount of your PCR reactions +3 more. Always prepare more, because of the errors of the pipettes.
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| - | b. Master mix should contain dNTPs, buffer and water.
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| - | Remember to put in following order:
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| - | 1. Master mix
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| - | 2. Primers
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| - | 3. Template DNA
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| - | 4. Enzyme
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| - | Remember primers should be diluted 1:10.
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| - | Phusion is used if we want to have better proof-reading.
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| - | In order to estimate size and amount of DNa fragments look below:
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| - | Estimating the size of sample:
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| - | We may look at the band intensities and from proportion and amount of DNA given in the picture below calculate the concentration of our DNA sample.??
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Latest revision as of 19:16, 21 September 2011
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