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Team:DTU-Denmark/PCR protocol
From 2011.igem.org
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
- Prepare master mix for the amount of your PCR reactions +3 more. Always prepare more, because of the errors of the pipettes.
- Master mix should contain what all reactions has in common
- Enzyme should be added last
Remember to add reagents in the following order
- Master mix
- (Primers)
- (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.
