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Team:DTU-Denmark/PCR protocol
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(Created page with "== 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 react...")
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Revision as of 14:44, 18 September 2011
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) 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 a. Prepare master mix for the amount of your PCR reactions +3 more. Always prepare more, because of the errors of the pipettes. b. Master mix should contain dNTPs, buffer and water. Remember to put in following order: 1. Master mix 2. Primers 3. Template DNA 4. Enzyme
Remember primers should be diluted 1:10. 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 size of sample: 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.??
