Team:DTU-Denmark/PCR protocol

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{{:Team:DTU-Denmark/Templates/Standard_page_begin|PCR protocol}}
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== PCR protocol ==
== PCR protocol ==
PCR reaction components:
PCR reaction components:
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== Estimating the amount of DNA in the sample: ==
== Estimating the amount of DNA in the sample: ==
By comparing the band intensities of the ladder you can estimate the concentration of DNA in your sample.
By comparing the band intensities of the ladder you can estimate the concentration of DNA in your sample.
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{{:Team:DTU-Denmark/Templates/Standard_page_end}}

Revision as of 15:04, 18 September 2011

PCR protocol

PCR protocol

PCR reaction components: a. Enzyme b. Forward primer c. Reverse primer d. dNTPs e. template DNA f. buffer g. MilliQ or distilled water

Amounts per one reaction (100μl)

Reagent TAQ + PFU [μl] Phusion [μl]
Enzyme 0.5 0.5
Forward primer 2.5 5
Reverse primer 2.5 5
dNTP 4 4
Template 1 1
Buffer 10 20
Water 79.5 64.5

Master mix

  1. Prepare master mix for the amount of your PCR reactions +3 more. Always prepare more, because of the errors of the pipettes.
  2. Master mix should contain what all reactions has in common
  3. Enzyme should be added last

Remember to add reagents in the following order

  1. Master mix
  2. (Primers)
  3. (Template DNA)

Remember primers should be diluted 1:10 from stock. Phusion is used if we want to have better proof-reading. In order to estimate size and amount of DNA fragments look below:

Estimating the amount of DNA in the sample:

By comparing the band intensities of the ladder you can estimate the concentration of DNA in your sample.