Team:DTU-Denmark/PCR protocol

From 2011.igem.org

Revision as of 14:44, 18 September 2011 by Lri (Talk | contribs)

(diff) ← Older revision | Latest revision (diff) | Newer revision → (diff)

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.??