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)

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.