Team:PKU Beijing/Notebook/Assembly/Haoqian Zhang

Notebook > Assembly > Haoqian Zhang's Note

2009.10.1
Positively transform 1-9C plasmid, intending to get pSB3T5 plasmid backbone.

One 5ml culture of LB medium and antibiotic (Tetracycline, 50ng/ml) was inoculated with a single positive colony from a LB agar plate. Cultures were grown in tubes for 14 hrs at 37°C with shaking at 70 rpm.

2009.10.2
Miniprep of the fresh culture containing 1-9C plasmid.

Digest 1-9C plasmid and T7p + RBS + CI plasmid with XbaI and PstI Double digestion system: Electrophoresis the digested samples, and extract the 1-9C plasmid backbone and T7p + RBS + CI insert.

2009.10.3
Ligate the T7p + RBS + CI insert to 1-9C plasmid backbone. System: Transform the ligation product, and plate on agar plate.

2009.10.4
Pick 3 colonies for each RBS, 18 colonies in total, incubated in LB medium with Tetracycline, 50ng/ml for 14 hrs.

Miniprep these 18 culture samples.

Digest the plasmids with XbaI and PstI to detect the successful ligation. Double digestion system: Electrophoresis the digested samples detect the successful ligation. The result showed that all the colonies did come from successful ligation.