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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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| - | {| class="wikitable"
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| - | |-
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| - | ! Reagent
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| - | ! TAQ + PFU [μl]
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| - | ! Phusion [μl]
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| - | |-
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| - | | Enzyme
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| - | | 0.5
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| - | | 0.5
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| - | |-
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| - | | Forward primer
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| - | | 2.5
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| - | | 5
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| - | |-
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| - | | Reverse primer
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| - | | 2.5
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| - | | 5
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| - | |-
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| - | | dNTP
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| - | | 4
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| - | | 4
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| - | |-
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| - | | Template
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| - | | 1
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| - | | 1
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| - | |-
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| - | | Buffer
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| - | | 10
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| - | | 20
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| - | |-
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| - | | Water
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| - | | 79.5
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| - | | 64.5
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| - | |}
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| - |
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| - | == Master mix ==
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| - | <ol style="list-style-type:lower-alpha">
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| - | <li> 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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| - | <li> Master mix should contain what all reactions has in common
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| - | <li> Enzyme should be added last
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| - | </ol>
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| - | Remember to add reagents in the following order
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| - | # Master mix
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| - | # (Primers)
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| - | # (Template DNA)
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| - |
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| - | Remember primers should be diluted 1:10 from stock.
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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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| - |
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| - | == Estimating the amount of DNA in the sample: ==
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| - | By comparing the band intensities of the ladder you can estimate the concentration of DNA in your sample.
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Latest revision as of 19:16, 21 September 2011
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