Pretend that you are doing a cloning. These are the steps:
1st step: A cloning vector (selective vector: β-Galactosidase/lac-Z) was cut with the restriction endonuclease Sma I, whose restriction site is:
C C C G G G
2nd step: The digested vector was treated with alkaline phosphatase to prevent the relegation of the plasmid.
3rd step: Your synthetic DNA of interest was inserted into the Sma I site in your vector.
Sequence of your fragment of interest:
5' G G A C T T A C T A C C C A A G T A 3'
3' C C T G A A T G A T G G G T T C A T 5'
Vector sequence indicating where the DNA fragment was inserted (Sma I site) in respect to the b-galactosidase gene (this is a blunt-end ligation!)
- ATG | ACC | ATG | ATT ACG | AAT | TCC | C GG | GGA -
↑
Sma I-cut
H3N- Met | Thr | Met | Ile | Thr | Asn | Ser | Arg | Gly --> functional b- galactosidase gene starts here
4th step: the digested vector and DNA fragment were ligated followed by a transformation into E. coli.
RESULTS: In the presence of the indicator X-gal, 52% of the colonies on the plate were blue and 48% were clear. The Blue colonies are not due to the re-ligation of the plasmid!
